Tymovirus virus and virus-like particles as nanocarriers for imaging and therapeutic agents

ABSTRACT

A method of targeting cancer tissue in a subject includes administering to the subject a plurality of functionalized Tymovirus virus or virus-like particles loaded with or conjugated to an imaging agent, a therapeutic agent or a targeting agent.

RELATED APPLICATION

This application claims priority from U.S. Provisional Application No.62/577,882, filed Oct. 27, 2017, the subject matter of which isincorporated herein by reference in its entirety.

GOVERNMENT FUNDING

This invention was made with government support under Grant Nos.R01-CA202814 awarded by The National Institutes of Health (NIH) and theNational Science Foundation (NSF). The United States government hascertain rights in the invention.

TECHNICAL FIELD

This application relates to methods and compositions for targetingcancer tissue in a subject and/or treating cancer in a subjectidentified as having cancer.

BACKGROUND

Nanocarrier platforms based on natural biological building blocks offernew opportunities in the biomedical and materials sciences. Viralnanoparticles (VNPs) are self-assembling supramolecular systems that canbe used to develop bioinspired nanomaterials and nanocarriers due totheir simple and inexpensive production, well-defined structuralfeatures, unique shapes and sizes, genetic programmability, and robustchemistries. VNPs based on plant viruses are particularly advantageousin medicine because they are biocompatible and biodegradable, but do notinfect humans and other mammals. They can carry drugs, imaging agents,and other nanoparticles in their internal cavity by assembly, infusion,or internal surface modification, and the external surface can bechemically or genetically engineered to attach targeting ligands fortissue-specific delivery. Plant VNPs have already overcome many of thechallenges of nanoparticle delivery, such as low stability in biologicalfluids, efficient delivery across membranes, avoidance of exocytosis,and targeting specificity. We and several others have established abroad range of plant VNPs such as those based on Cowpea mosaic virus(CPMV), Cowpea chlorotic mottle virus (CCMV), Brome mosaic virus (BMV),Potato virus X (PVX) and Tobacco mosaic virus (TMV).

Virus-like particles (VLPs) are a subset of VNPs, which lack the viralgenome and assemble spontaneously from virus structural proteins intononinfectious protein cage-like structures. Many different virusstructural proteins form VLPs when expressed in standard heterologousexpression systems such as Escherichia coli, yeast, plants, mammaliancells, and insect cells. Such VLPs tend to be structurally andmorphologically similar to the wildtype virus particles formed in vitroand demonstrate similar cell tropism, uptake, and intracellulartrafficking.

SUMMARY

Embodiments described herein relate to a method of targeting cancertissue in subject. The method includes administering to the subject aplurality of functionalized Tymovirus virus or Tymovirus virus-likeparticles (VLPs). The Tymovirus virus or VLPs can be administered to thesubject at an effective amount. The Tymovirus virus can belong to thephysalis mottle virus (PhMV) species. The Tymovirus virus or VLPs areloaded with or conjugated to one or more of a therapeutic agent, animaging agent, or a targeting agent. The targeted cancer tissue caninclude prostate, breast or ovarian cancer tissue. In some embodiments,the Tymovirus virus or VLPs have been PEGylated.

In some embodiments, the Tymovirus virus or VLPs include an imagingagent. The imaging agent can include a fluorescent molecule forfluorescent imaging or a chelated metal. In some embodiments, the methodcan further include the step of imaging cancer tissue in the subjectusing an imaging device subsequent to administering the Tymovirus virusor VLPs.

In some embodiments, the Tymovirus virus or VLPs include a therapeuticagent (e.g., a cytotoxic compound). The therapeutic agent can include anantitumor agent such as doxorubicin or mitoxantrone. In someembodiments, the therapeutic agent can include a photodynamictherapeutic (PDT) photosensitizer agent. The PDT agent can be selectedfrom a porphyrin or a mettalloporphyrin compound. In certainembodiments, the PDT agent is a cationic zinc ethynylphenyl porphyrin.

In some embodiments, the one or more of a therapeutic agent, an imagingagent, or a targeting agent is directly conjugated to the Tymovirusvirus or VLPs. In some embodiments, the one or more of a therapeuticagent, an imaging agent, or a targeting agent is conjugated to theTymovirus virus or VLPs particles via a linker.

In some embodiments, the Tymovirus virus or VLPs include multipletargeting agents. The spacing and location of the targeting agents onthe Tymovirus virus or VLPs can be controlled to facilitate delivery,targeting, and/or therapeutic efficacy of the Tymovirus virus or VLPswhen administered to a subject.

Other embodiments described herein relate to a method of treating cancerin a subject in need thereof. The method includes administering to thesubject a therapeutically effective amount of a plurality offunctionalized Tymovirus virus or VLPs loaded with or conjugated to oneor more therapeutic agents. The Tymovirus virus or VLPs can beadministered together with a pharmaceutically acceptable carrier. Thecancer treated can include prostate, breast or ovarian cancer. TheTymovirus virus can belong to the physalis mottle virus (PhMV) species.In some embodiments, the Tymovirus virus or VLPs have been PEGylated.

In some embodiments, the therapeutic agent includes a cytotoxiccompound. The cytotoxic compound can include an antitumor agent such asdoxorubicin or mitoxantrone.

In some embodiments, the therapeutic agent includes a photodynamictherapeutic (PDT) photosensitizer agent. The PDT agent can be selectedfrom a porphyrin or a mettalloporphyrin compound. In certainembodiments, the PDT agent is a cationic zinc ethynylphenyl porphyrin.

In some embodiments, the Tymovirus virus or VLPs further include one ormore targeting agents. In some embodiments, the targeting agents can beconjugated to the Tymovirus virus or VLPs. The targeting agents can bedirectly conjugated to the Tymovirus virus or VLPs or conjugated to theTymovirus virus or VLPs via a linker.

In some embodiments, the Tymovirus virus or VLPs include multipletargeting agents. The spacing and location of the targeting agents onthe Tymovirus virus or VLPs can be controlled to facilitate delivery,targeting, and/or therapeutic efficacy of the Tymovirus virus or VLPswhen administered to a subject.

Another embodiment described herein relate to a method of detectingcancer in a subject. The method includes administering to the subject aplurality of functionalized Tymovirus virus or Tymovirus virus-likeparticles (VLPs) that have been loaded with or conjugated to an imagingagent. The method also includes detecting the imaging agent in thesubject using an imaging device subsequent to administering theTymovirus virus or VLPs to determine the location and/or distribution ofthe cancer in the subject. The Tymovirus virus or VLPs can beadministered together with a pharmaceutically acceptable carrier.

The detected cancer can include breast cancer, ovarian cancer, orprostate cancer. The Tymovirus virus can belong to the physalis mottlevirus (PhMV) species. In some embodiments, the Tymovirus virus or VLPshave been PEGylated.

In some embodiments, the imaging agent can include a fluorescentmolecule for fluorescent imaging or a chelated metal for MRI imaging.

In some embodiments, the Tymovirus virus or VLPs further include one ormore targeting agents. In some embodiments, the targeting agents can beconjugated to the Tymovirus virus or VLPs. The targeting agents can bedirectly conjugated to Tymovirus virus or VLPs or conjugated to theTymovirus virus or VLPs via a linker.

In some embodiments, the Tymovirus virus or VLPs include multipletargeting agents. The spacing and location of the targeting agents onthe Tymovirus virus or VLPs can be controlled to facilitate delivery,targeting, and/or therapeutic efficacy of the Tymovirus virus or VLPswhen administered to a subject.

Other embodiments described herein relate to a functionalized Tymovirusbased nanoparticle. The nanoparticle includes a Tymovirus virus orTymovirus virus-like particle (VLP) that has been loaded with orconjugated to one or more of an imaging agent, a therapeutic agent, or atargeting agent. The Tymovirus virus can belong to the physalis mottlevirus (PhMV) species. In some embodiments, the Tymovirus virus or VLPhas been PEGylated.

In some embodiments, the imaging agent, the therapeutic agent, or thetargeting agent is conjugated directly or indirectly to the interior ofthe Tymovirus virus or VLP. In some embodiments, the imaging agent, thetherapeutic agent, or the targeting agent is conjugated directly orindirectly via a linker to the exterior of the Tymovirus virus or VLP.In other embodiments, the imaging agent, the photodynamic therapeutic(PDT) photosensitizer agent or the cytotoxic compound is non-covalentlyinfused into the interior of the Tymovirus virus or VLP.

In some embodiments, the Tymovirus virus or VLP includes an imagingagent. The imaging agent can include a fluorescent molecule forfluorescent imaging or a chelated metal for MRI imaging.

In some embodiments, the Tymovirus virus or VLP includes a therapeuticagent, such as a cytotoxic compound. The therapeutic agent can includean antitumor agent such as doxorubicin or mitoxantrone.

In some embodiments, the therapeutic agent includes a photodynamictherapeutic (PDT) photosensitizer agent. The PDT agent can be selectedfrom a porphyrin or a mettalloporphyrin compound. In certainembodiments, the PDT agent is a cationic zinc ethynylphenyl porphyrin.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1(A-E) illustrate a structure of PhMV and the strategies used forthe functionalization of PhMV-derived VLPs. (A) Ribbon diagram of thePhMV VLP icosahedral asymmetric unit consisting of A, B and C subunits.Five A subunits make up pentameric capsomeres at the icosahedral 5-foldaxes, while B and C subunits form hexamers at the icosahedral 3foldaxes. Representation of internal and external surfaces (with 180-degreerotation) of the PhMV asymmetric unit, highlighting surface-exposed(K62, K143, K153 and K166) and buried (K8, K10, K76, K182 and K127)lysine residues (blue), and the single cysteine residue (C75, green).PhMV forms from 180 identical coat protein subunits arranged in a T=3icosahedral structure. Images created using UCSF Chimera (PDB: 1E57).The capsid is characterized by prominent protrusions of pentamers andhexamers. (B) Schematic of PhMV labeling with sulfo-Cy5 NHS ester usinglysine-NHS ester chemistry. (C) Conjugation of Cy5.5-maleimide tointernal cysteine residues using maleimide-thiol chemistry. (D) DOXinfusion into PhMV, leading to cargo-loaded particles. Washing andultracentrifugation is used to remove excess DOX, yielding intact PhMVwith infused DOX (DOX-PhMV). (E) Schematic of PS (blue) loading intoPhMV via infusion, yielding PS-PhMV particles.

FIGS. 2(A-D) illustrate characterization of fluorophore-labeled anddrug-loaded VLPs. (A) SDS-PAGE analysis of PhMV-KECyS, PhMV-CI-Cy5.5,PS-PhMV, and DOX-PhMV visualized under UV light (UV), white light (WL)before staining, and under white light after Coomassie blue staining(CS). M=SeeBlue Plus2 molecular weight (kDa) standard; 1=Native PhMV;2=PhMV-KE-Cy5; 3=PhMV-CI-Cy5.5; 4=PS-PhMV; 5=PS; 6=DOX-PhMV; 7=DOX. (B)Agarose gel electrophoresis of PhMV-KE-Cy5, PhMV—CI-Cy5.5, PS-PhMV, andDOX-PhMV visualized under UV light and white light before, and underwhite light after Coomassie blue staining (CS). Functionalized particlesloaded in each lane was same as described above for SDS-PAGE analysis(note: the white marks in the center of the gel are the pockets intowhich the samples were loaded prior to electrophoretic separation). (C)Size exclusion chromatograms of PhMV-KE-Cy5 [monitored at 260 nm (blue),280 nm (red) and 646 nm (green, sulfo-Cy5 NHS ester absorbance],PhMV-CI-Cy5.5 [monitored at 260 nm (blue), 280 nm (red) and 673 nm(green, Cy5.5-maleimide absorbance], PS-PhMV [monitored at 260 nm(blue), 280 nm (red) and 450 nm (green, PS absorbance] and DOX-PhMV[monitored at 260 nm (blue), 280 nm (red) and 496 nm (green, DOXabsorbance]. (D) Transmission electron micrographs of negatively stained(UAc) PhMV-KE-Cy5, PhMV-CI-Cy5.5, PS-PhMV, and DOX-PhMV.

FIGS. 3(A-D) illustrate the characterization of PhMV-biotin conjugates.(A) Biotinylated PhMV particles separated by denaturing SDS-PAGEvisualized after staining with Coomassie. M=SeeBlue Plus2 molecularweight marker. 1. Native PhMV; 2. PhMV-CI-bio; 3. PhMV-KE-bio. (B)Biotinylated PhMV particles separated by agarose gel electrophoresisvisualized after Coomassie staining. (C) Flow through and elutedbiotinylated particles from avidin bead binding assay separated bySDS-PAGE and stained with Coomassie. 4. Native PhMV flow through; 5.PhMV-KE-bio flow through; 6. PhMV-CI-bio flow through; 7. Bound nativePhMV; 8. Bound PhMV-KE-bio; 9. Bound PhMV-CI-bio. (D) Avidin bead assay:PhMV samples are exposed to avidin-coated beads; only particles withbiotin on the external surface bind to the beads.

FIGS. 4(A-D) illustrate cell uptake studies with fluorescence-labeledPhMV using confocal microscopy and FACS. (A) Confocal imagesrepresenting the internalization of PhMV-KE-Cy5 in A2780, MDA-MB-231 andPC-3 cells. PhMV was tagged with sulfo-Cy5 NHS ester (pseudo green), thecell membrane was stained with wheat germ agglutinin (WGA)-Alexa Fluor555 (pseudo pink) and the nucleus was stained with DAPI (blue). Scalebars=25 μm (B) Flow cytometry of A2780, MDA-MB-231 and PC-3 cellsfollowing 6 h incubation with PhMV-K_(E)-Cy5 particles. Percentage ofpositive cells for each sample was quantified from three replicates andrepresented with standard deviation (±) in the corresponding cellpanels. (C) Confocal imaging of A2780 and MDA-MB-231 cells showingcolocalization of PhMV-C_(I)-Cy5.5 particles with the endolysosomalmarker LAMP-1 after 6 h. Nuclei are shown in blue, endolysosomes arestained with mouse anti-human LAMP-1 antibody (red) and PhMV-C_(I)-Cy5.5(pseudo green). Colocalization signals are shown in white (overlay,bottom panel). Scale bars=25 μm. (D) FACS quantification ofPhMV-C_(I)-Cy5.5 uptake using A2780, MDA-MB-231, PC-3, HeLa, RAW 264.7,U87, HT1080 and NIH/3T3 cells. All samples were measured in triplicatesand analyzed using FlowJo software.

FIG. 5 illustrates the number of dye/drug (guest) molecules loaded perVLP via infusion at different molar excesses (averaged data from 2experiments are shown). The number of guest molecules per particle wasdetermined by UV/vis absorbance and the Bradford assay was used todetermine the protein concentration. The chemical structure of eachguest molecule is depicted with their respective charge. The amine groupof DOX is annotated with asterisks to indicate a site of protonation(positive charge) in physiological conditions.

FIGS. 6(A-D) illustrate evaluation of cytotoxic efficacy of drug-loadedPhMV particles. (A) MTT cell viability assay of PC-3 cells usingPS-PhMV. Cell viability was measured following 8 h incubation withvarying concentrations of PS or PS-PhMV and 30 min illumination withwhite light (no cell killing was observed when cells were incubated inthe dark, not shown). (B) LIVE/DEAD assay of PC-3 cells showingrepresentative images after photodynamic therapy of cells incubated withPS-PhMV or free PS and LIVE/DEAD cell staining. Calcein-AM staining oflive cells and ethidium homodimer-1 staining is shown. Scale bar=100 μm.Illuminated cells incubated with PS-PhMV showed a slight increase incell killing efficacy (IC₅₀=0.03 μM) compared to free PS (IC₅₀=0.05 μM).Dark controls show no cytotoxicity with PS-PhMV or PS. Scale bar=100 μm.(C). Efficacy of DOX-PhMV versus DOX using A2780 (human ovarian cancer)and (D) MDA-MB-231 (human breast cancer) cells as determined by MTTassay. Cells were treated with DOX or DOX-PhMV corresponding to 0, 0.01,0.05, 0.1, 0.5, 1, 5 and 10 μM for 24 h. IC₅₀ values were determinedusing GraphPad Prism software.

DETAILED DESCRIPTION

Methods involving conventional molecular biology techniques aredescribed herein. Such techniques are generally known in the art and aredescribed in detail in methodology treatises, such as Current Protocolsin Molecular Biology, ed. Ausubel et al., Greene Publishing andWiley-Interscience, New York, 1992 (with periodic updates). Unlessotherwise defined, all technical terms used herein have the same meaningas commonly understood by one of ordinary skill in the art to which theapplication pertains. Commonly understood definitions of molecularbiology terms can be found in, for example, Rieger et al., Glossary ofGenetics: Classical and Molecular, 5th Edition, Springer-Verlag: NewYork, 1991, and Lewin, Genes V, Oxford University Press: New York, 1994.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. The terminology used in thedescription of the invention herein is for describing particularembodiments only and is not intended to be limiting of the invention.All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety.

Definitions

As used in this specification and the appended claims, the singularforms “a”, “an” and “the” include plural references unless the contentclearly dictates otherwise. Thus, for example, reference to “a cell”includes a combination of two or more cells, and the like.

The term “about” as used herein when referring to a measurable valuesuch as an amount, a temporal duration, and the like, is meant toencompass variations of ±20% or 110%, more preferably ±5%, even morepreferably ±1%, and still more preferably ±0.1% from the specifiedvalue, as such variations are appropriate to perform the disclosedmethods.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice for testing of the present invention, the preferredmaterials and methods are described herein. In describing and claimingthe present invention, the following terminology will be used.

“Image” or “imaging” refers to a procedure that produces a picture of anarea of the body, for example, organs, bones, tissues, cells or blood.

“Treat”, “treating”, and “treatment”, etc., as used herein, refer to anyaction providing a benefit to a subject afflicted with a condition ordisease such as cancer, including improvement in the condition throughlessening or suppression of at least one symptom, delay in progressionof the disease, etc.

Prevention, as used herein, refers to any action providing a benefit toa subject at risk of being afflicted with a condition or disease such ascancer, including avoidance of the development of cancer or a decreaseof one or more symptoms of the disease should cancer develop. Thesubject may be at risk due to exposure to a carcinogen, or as a resultof family history.

A “subject,” as used herein, can be any animal, and may also be referredto as the patient. Preferably the subject is a vertebrate animal, andmore preferably the subject is a mammal, such as a domesticated farmanimal (e.g., cow, horse, pig) or pet (e.g., dog, cat). In someembodiments, the subject is a human.

“Pharmaceutically acceptable” as used herein means that the compound orcomposition is suitable for administration to a subject for the methodsdescribed herein, without unduly deleterious side effects in light ofthe severity of the disease and necessity of the treatment.

The terms “therapeutically effective” and “pharmacologically effective”are intended to qualify the amount of each agent which will achieve thegoal of decreasing disease severity while avoiding adverse side effectssuch as those typically associated with alternative therapies. Thetherapeutically effective amount may be administered in one or moredoses.

“Targeting,” as used herein, refers to the ability of modifiedvirus-like particles to be delivered to and preferentially accumulate incancer tissue in a subject compared to normal tissue.

As used herein, the term “targeting agent” can refer to a molecule ormolecules that are able to bind to and complex with a biomarker. Theterm can also refer to a functional group that serves to target ordirect a nanoparticle, therapeutic agent or anti-cancer agent to aparticular location, cell type, diseased tissue, or association. Ingeneral, a “targeting agent” can be directed against a biomarker.

As used herein, the term “molecular signature” can refer to a uniqueexpression pattern of one or more biomarkers (e.g., gene(s) orprotein(s)) of a cell.

As used herein, the term “antibody” refers to an immunoglobulin,derivatives thereof which maintain specific binding ability, andproteins having a binding domain which is homologous or largelyhomologous to an immunoglobulin binding domain. These proteins may bederived from natural sources, or partly or wholly syntheticallyproduced. An antibody may be monoclonal or polyclonal. The antibody maybe a member of any immunoglobulin class, including any of the humanclasses: IgG, IgM, IgA, IgD, and IgE. In exemplary embodiments,antibodies used with the methods and compositions described herein arederivatives of the IgG class.

As used herein, the term “antibody fragment” refers to any derivative ofan antibody which is less than full-length. In exemplary embodiments,the antibody fragment retains at least a significant portion of thefull-length antibody's specific binding ability. Examples of antibodyfragments include, but are not limited to, Fab, Fab′, F(ab′)₂, scFv, Fv,dsFv diabody, and Fd fragments. The antibody fragment may be produced byany means. For instance, the antibody fragment may be enzymatically orchemically produced by fragmentation of an intact antibody, it may berecombinantly produced from a gene encoding the partial antibodysequence, or it may be wholly or partially synthetically produced. Theantibody fragment may optionally be a single chain antibody fragment.Alternatively, the fragment may comprise multiple chains which arelinked together, for instance, by disulfide linkages. The fragment mayalso optionally be a multimolecular complex. A functional antibodyfragment will typically comprise at least about 10 amino acids and moretypically will comprise at least about 200 amino acids.

As used herein, the term “diabodies” refers to dimeric scFvs. Thecomponents of diabodies typically have shorter peptide linkers than mostscFvs and they show a preference for associating as dimers.

As used herein, the term “epitope” refers to a physical structure on amolecule that interacts with a selective component. In exemplaryembodiments, epitope refers to a desired region on a target moleculethat specifically interacts with a selectivity component.

As used herein, the term “Fab′” refers to an antibody fragment that isessentially equivalent to that obtained by reduction of the disulfidebridge or bridges joining the two heavy chain pieces in the F(ab′)₂fragment. Such fragments may be enzymatically or chemically produced byfragmentation of an intact antibody, recombinantly produced from a geneencoding the partial antibody sequence, or it may be wholly or partiallysynthetically produced.

As used herein, the term “F(ab′)₂” refers to an antibody fragment thatis essentially equivalent to a fragment obtained by digestion of animmunoglobulin (typically IgG) with the enzyme pepsin at pH 4.0-4.5.Such fragments may be enzymatically or chemically produced byfragmentation of an intact antibody, recombinantly produced from a geneencoding the partial antibody sequence, or it may be wholly or partiallysynthetically produced.

As used herein, the term “Fv” refers to an antibody fragment thatconsists of one V_(H) and one V_(L) domain held together by noncovalentinteractions. The term “dsFv” is used herein to refer to an Fv with anengineered intermolecular disulfide bond to stabilize the V_(H)—V_(L)pair.

As used herein, the term “immunogen” traditionally refers to compoundsthat are used to elicit an immune response in an animal, and is used assuch herein. However, many techniques used to produce a desiredselectivity component, such as the phage display and aptamer methodsdescribed below, do not rely wholly, or even in part, on animalimmunizations. Nevertheless, these methods use compounds containing an“epitope,” as defined above, to select for and clonally expand apopulation of selectivity components specific to the “epitope.” These invitro methods mimic the selection and clonal expansion of immune cellsin vivo, and, therefore, the compounds containing the “epitope” that isused to clonally expand a desired population of phage, aptamers and thelike in vitro are embraced within the definition of “immunogens.”

As used herein, the terms “single-chain Fvs” and “scFvs” refers torecombinant antibody fragments consisting of only the variable lightchain (V_(L)) and variable heavy chain (V_(H)) covalently connected toone another by a polypeptide linker. Either V_(L) or V_(H) may be theNH₂-terminal domain. The polypeptide linker may be of variable lengthand composition so long as the two variable domains are bridged withoutserious steric interference. In exemplary embodiments, the linkers arecomprised primarily of stretches of glycine and serine residues withsome glutamic acid or lysine residues interspersed for solubility.

Embodiments described herein relate to Virus-like particles (VLPs)derived from the Tymovirus genus of plant viruses, such as physalismottle virus (PhMV), for use as platforms for diagnostics andtherapeutics by functionalizing Tymovirus based VLPs with drugs,targeting and/or imaging molecules.

The VLP platforms described herein may be engineered and tailored fordesired functional applications through genetic modification,non-covalent infusion and/or bioconjugate chemistry. Protein engineeringcan be used to introduce new functionalities at three distinctinterfaces of VLPs: internal, external, and inter-subunit. This allowsthe fine tuning of surface charge, drug encapsulation, ligand display,and particle stability.

In order to functionalize Tymovirus based VLPs, multiple approaches,such as bioconjugation chemistries and non covalent infusion protocolsare described herein that can be used to modify the behavior andproperties of the VLPs. Once functionalized, Tymovirus based VLPnanoparticles can be characterized by flow cytometry and confocalmicroscopy and their cytotoxic efficacy have been evaluated in humannormal and ovarian, breast and prostate cancer cell lines as well as inex vivo organ biodistribution analysis models, and in an in vivo mousemodels of prostate cancer.

In one aspect, the invention provides a method of using a Tymovirusvirus or Tymovirus virus-like particle (VLP) nanoparticle to targetcancer tissue in a subject. The use of a Tymovirus virus or VLPs allowsfor nanoparticles that are biocompatible, biodegradeable, non-infectiousin mammals. In addition, the Tymovirus virus or VLPs can readily bechemically engineered to carry cargo such as a therapeutic agent, animaging agent, or a targeting agent. The method includes administering aplurality of functionalized Tymovirus virus or Tymovirus virus-likeparticles (VLPs) loaded with or conjugated to one or more or atherapeutic agent, an imaging agent, or a targeting agent to thesubject. As defined herein, targeting cancer tissue refers to theability of the Tymovirus virus or Tymovirus VLPs to reach and preferablyaccumulate within cancer tissue after being administered to the subject.The ability of Tymovirus virus or VLPs to target cancer tissue issupported by the characterization, cytotoxicity, biodistribution, tumormodel studies described herein.

The Tymovirus VLPs are shown to colocalize with the endolysosomal markerLAMP-1 within about 6 hours of administration to cancer cells. While notintending to be bound by theory, it is believed that Tymovirus virus orTymovirus VLPs are preferentially internalized by cancer cells overnon-cancerous cells via endocytosis, thereby delivering thefunctionalized VLPs to the tumor cells at a much higher efficiency thannon-transformed cells. Embodiments of the invention can deliver andinternalize about 10%, about 20%, about 30%, about 40%, about 50%, about60%, or even about 70% or more of administered externally functionalizedVLPs to a subject's cancer cells. Embodiments of the invention candeliver and internalize about 10%, about 20%, about 30%, about 40%,about 50%, about 60%, about 70%, about 80%, about 90% or even about 100%of administered internally functionalized virus or VLPs to a subject'scancer cells. In specific embodiments, the Tymovirus virus or TymovirusVLPs described herein can deliver and internalize about 95% to about100% of administered internally functionalized Tymovirus virus orTymovirus VLPs to cancer cells.

Tymoviruses

Embodiments described herein can include a functionalized Tymovirusvirus nanoparticle. Other embodiments can relate to functionalizedTymovirus virus-like particles (VLPs) where the Tymovirus VLPs arederived from a virus of the Tymovirus genus. Tymovirus virus is a virusthat primarily infects plants and has a non-enveloped icosahedral andisometric structure. The diameter of a Tymovirus, such as PhMV, is about30 nm. Use of a Tymovirus virus or Tymovirus VLP as described hereinprovides the advantages of improved physical stability (e.g., aftercargo loading as well as in storage) and production consistency.

A Tymovirus virus can be selected from a group consisting of PhysalisMottle Virus (PhMV), Belladonna Mottle Virus, Turnip Yellow MosaicVirus, Cacao Yellow Mosaic Virus, Clitoria Yellow Vein Virus, DesmodiumYellow Mottle Virus, Eggplant Mosiac Virus and Passion Fruit YellowMosaic Virus. A comparison of coat protein sequence of PhMV with othertymoviruses revealed that PhMV has a 52% identity with belladonna mottlevirus (E) and 33% identity with turnip yellow mosaic virus (TYMV),showing that PhMV (previously named as belladonna mottle virus 1) is adistinct Tymovirus. Thus, in certain embodiments, the Tymovirus virusand the Tymovirus VLP can be derived from PhMV.

PhMV is a small spherical plant virus of the Tymovirus genus ofpositive-stranded RNA viruses. The nucleotide sequence coding for the(PhMV) coat protein was identified from the GenBank having EMBLaccession number S97776 (Jocob et al., 1992). The positive-sense RNAgenome is encapsidated in a protein shell consisting of 180 identicalcopies of coat protein (CP) arranged with T=3 icosahedral symmetry. Themultiple copies of the asymmetric unit provide regularly spacedattachment sites on both the internal and external surfaces of the PhMVcapsid allowing for modification of PhMV with diagnostic and therapeuticagents described herein.

The coat protein of a Tyrovirus virus for use as a VLP can besynthetically produced using methods well known in the art. Methods ofproducing Tymovirus VLPs can include the steps of: (a) producing arecombinant polynucleotide sequence, (b) constructing a recombinantvector comprising a regulatory sequence and the recombinantpolynucleotide sequence of step (a), (c) transforming a host cell withthe recombinant vector of step (b) to produce a recombinant host cell,(d) growing the recombinant host cell of step (c) to produce Tymovirusvirus-like particles, and (e) purifying the Tymovirus virus-likeparticles of step (d). The recombinant vector can further include aregulatory sequence. Exemplary regulatory sequence can include T7, SP6and T3 promoters.

In an exemplary embodiment, Tymovirus-derived VLPs can be formed fromTymovirus structural proteins encoded by a recombinant poly nucleotidesequence that are expressed in an Escherichia coli, yeast or baculovirusheterologous expression system. In some embodiments, the heterologousexpression system is an E. coli expression system. The E. coli straincan be selected from the group consisting of JM101, DH5α, BL21, HB101,BL21(DE3) pLys S, XL-1 Blue and Rossetta. In some embodiments, therecombinant poly nucleotide sequence can include, for example, anucleotide sequence encoding all, or a truncated portion, of the PhMVcoat protein.

Tymovirus Virus or Tymovirus VLP Functionalization

The invention makes use of a functionalized Tymovirus virus or TymovirusVLP that have been loaded with or conjugated to one or more of atherapeutic agent, an imaging agent, or a targeting agent. Including atherapeutic agent, an imaging agent, or a targeting agent provides thecapability for the virus particle to function as a targeted imagingagent or a targeted therapeutic agent. The ability of a Tymovirus virusor Tymovirus VLP to preferentially target cancer tissue can be furtherenhanced by loading or conjugating a targeting agent to the Tymovirusvirus or Tymovirus VLP.

In some embodiments, therapeutic agents, imaging agents, and/ortargeting agents (collectively referred to herein as agents) can beconjugated to the Tymovirus virus or Tymovirus VLP by any suitabletechnique, with appropriate consideration of the need forpharmacokinetic stability and reduced overall toxicity to the patient.The term “conjugating” when made in reference to an agent and aTymovirus virus or Tymovirus VLP as used herein means covalently linkingthe agent to the virus. In certain embodiments, the nature and size ofthe agent and the site at which it is covalently linked to the virusparticle do not interfere with the biodistribution of the modified virusand/or interfere with the internalization of the Tymovirus virus orTymovirus VLPs by cancer cells.

Because viral capsids are proteinaceous, standard bioconjugationprotocols that address chemically reactive amino acid side chains can beused as with other proteins. The most common reactions used to modifyviruses involve the reactive side chains of lysine, cysteine andaspartic/glutamic acid residues, which are accessible toN-hydroxysuccinimidyl (NHS) chemistry, Michael addition to maleimides,and carbodiimide activation, respectively.

An agent can be conjugated to a Tymovirus virus or Tymovirus VLP eitherdirectly or indirectly (e.g. via a linker group). In some embodiments,the agent is directly attached to a functional group capable of reactingwith the agent. For example, viral coat proteins include lysines thathave a free amino group that can be capable of reacting with acarbonyl-containing group, such as an anhydride or an acid halide, orwith an alkyl group containing a good leaving group (e.g., a halide).Viral coat proteins also contain glutamic and aspartic acids. Thecarboxylate groups of these amino acids also present attractive targetsfor functionalization using carbodiimide activated linker molecules;cysteines can also be present which facilitate chemical coupling viathiol-selective chemistry (e.g., maleimide-activated compounds). Inaddition, genetic modification can be applied to introduce any desiredfunctional residue, including non-natural amino acids, e.g. alkyne- orazide-functional groups. See Pokorski, J. K. and N. F. Steinmetz MolPharm 8(1): 29-43 (2011).

Alternatively, a suitable chemical linker group can be used. A linkergroup can serve to increase the chemical reactivity of a substituent oneither the agent or the virus particle, and thus increase the couplingefficiency. A preferred group suitable for attaching agents to theTymovirus virus particle or VLP are lysine residues present in the viralcoat protein.

Suitable linkage chemistries include maleimidyl linkers and alkyl halidelinkers and succinimidyl (e.g., N-hydroxysuccinimidyl (NHS)) linkers(which react with a primary amine on the plant virus particle). Severalprimary amine and sulfhydryl groups are present on viral coat proteins,and additional groups can be designed into recombinant viral coatproteins. It will be evident to those skilled in the art that a varietyof bifunctional or polyfunctional reagents, both homo- andhetero-functional (such as those described in the catalog of the PierceChemical Co., Rockford, Ill.), can be employed as a linker group.Coupling can be effected, for example, through amino groups, carboxylgroups, sulfhydryl groups or oxidized carbohydrate residues.

Other types of linking chemistries are also available. For example,methods for conjugating polysaccharides to peptides are exemplified by,but not limited to coupling via alpha- or epsilon-amino groups toNaIO₄-activated oligosaccharide (Bocher et al., J. Immunol. Methods 27,191-202 (1997)), using squaric acid diester(1,2-diethoxycyclobutene-3,4-dione) as a coupling reagent (Tietze et al.Bioconjug Chem. 2:148-153 (1991)), coupling via a peptide linker whereinthe polysaccharide has a reducing terminal and is free of carboxylgroups (U.S. Pat. No. 5,342,770), and coupling with a synthetic peptidecarrier derived from human heat shock protein hsp65 (U.S. Pat. No.5,736,146). Further methods for conjugating polysaccharides, proteins,and lipids to plant virus peptides are described by U.S. Pat. No.7,666,624.

In some embodiments, it can be desirable to use a linker group which iscleavable during or upon internalization into a cell, or which isgradually cleavable over time in the extracellular environment. A numberof different cleavable linker groups have been described. The mechanismsfor the intracellular release of a cytotoxic agent from these linkergroups include cleavage by reduction of a disulfide bond (e.g., U.S.Pat. No. 4,489,710); by irradiation of a photolabile bond (e.g., U.S.Pat. No. 4,625,014); by hydrolysis of derivatized amino acid side chains(e.g., U.S. Pat. No. 4,638,045); by serum complement-mediated hydrolysis(e.g., U.S. Pat. No. 4,671,958); and acid-catalyzed hydrolysis (e.g.,U.S. Pat. No. 4,569,789).

In some embodiments, more than one of a therapeutic agent, an imagingagent, or a targeting agent can be conjugated to a Tymovirus virus orTymovirus VLP of the invention. By poly-derivatizing the Tymovirus virusor Tymovirus VLPs of the invention, several therapeutic (e.g.,cytotoxic) strategies can be simultaneously implemented. For example, aplurality of Tymovirus virus or Tymovirus VLPs can be made useful as acontrasting agent for several visualization techniques, or a Tymovirusvirus or Tymovirus VLP including a therapeutic agent can be labeled fortracking by a visualization technique. In one embodiment, multiplemolecules of a therapeutic agent, an imaging agent, or a targeting agentare conjugated to a Tymovirus virus or Tymovirus VLP. In anotherembodiment, more than one type of a therapeutic agent, an imaging agent,or a targeting agent can be conjugated to a Tymovirus virus or TymovirusVLP.

Non-Covalent Infusion of Imaging Agents and Cytotoxic Compounds

In some embodiments, Tymovirus virus or Tymovirus VLPs can befunctionalized by loading with or conjugated to a therapeutic agent, animaging agent, or a targeting agent through the use of non-covalentinfusion techniques that facilitate efficient cargo loading of one ormore of a therapeutic agent, an imaging agent, or a targeting agent intothe virus or VLPs (See FIG. 1D, E). The three dimensional crystalstructure of empty Tymovirus VLPs (e.g., PhMV-derived VLPs) correspondto a “swollen state” of the virus capable of cargo loaded vianon-covalent infusion. To load cargo into Tymovirus virus or TymovirusVLPs, Tymovirus virus or Tymovirus VLPs can be incubated in a bathingsolution containing the guest molecule(s) (e.g., therapeutic agent,imaging agent, and/or targeting agent) at a molar excesses ranging fromabout 100 to about 10,000 molecules per VLP) in KP buffer with 10% (v/v)DMSO overnight at room temperature. After the reaction, excess guestmolecules can be removed by ultracentrifugation and the amount ofprotein and cargo can be quantified by the Bradford assay and UV/visiblespectroscopy, respectively.

It was found using TEM analysis that the Tymovirus VLP loaded with acargo agent via non-covalent infusion maintain the approximatelyspherical structure of a wild-type particle and that there was nosignificant change in particles diameter (see FIG. 2D). It was furthershown using fast protein liquid chromatography (FPLC) that the stabilityof the loaded VLPs can remain stable after months of storage in KPbuffer at 4° C. with no evidence of particle aggregation.

Differences in loading efficiency may reflect the density anddistribution of charged and hydrophobic groups on the guest molecules(e.g., a cytotoxic agent). For example, Tymovirus virus or TymovirusVLPs typically have a greater affinity for cargo having a positivecharge. Thus, in some embodiments, Tymovirus virus or Tymovirus VLPs areloaded with one or more positively charged therapeutic agents, imagingagents and/or targeting agents. In certain embodiments, cargo agentloaded PhMV is taken up by endocytosis and is trafficked to the lysosomewhere the protein carrier is degraded thus releasing the guest molecule(e.g., a cytotoxic agent) which diffuses into the cytosol and in thecase of a cytotoxic agent, can kill the cells.

Imaging Agents

In some embodiments, the functionalized Tymovirus virus or TymovirusVLPs are loaded with or conjugated to one or more imaging agents; i.e.,the Tymovirus VLP comprises an imaging agent. Examples of imaging agentsinclude fluorescent, radioactive isotopes, MRI contrast agents,enzymatic moieties, or detectable label of the invention. In someembodiments, the imaging agent is a fluorescent molecule for fluorescentimaging allowing, for example, quantification by UV/visible spectroscopybased on absorbance. In some embodiments, the imaging agent is a MRIcontrast agent such as a chelated metal (e.g., Gd, Tb, or Dy).

The detectable group can be any material having a detectable physical orchemical property. Such detectable labels have been well-developed inthe field of fluorescent imaging, magnetic resonance imaging, positiveemission tomography, or immunoassays and, in general, most any labeluseful in such methods can be applied to the present invention. Thus, alabel is any composition detectable by spectroscopic, photochemical,biochemical, immunochemical, electrical, optical or chemical means.Useful labels in the present invention include magnetic beads (e.g.Dynabeads™), a triarylmethane dye (e.g., crystal violet), fluorescentdyes (e.g., fluorescein isothiocyanate, cyanines such as Cy5, Cy5.5 andanalogs thereof (e.g., sulfo-Cyanine 5 NHS ester and Cy5.5 maleimide),Alexa Fluor dye (e.g., Alexa Fluor 647 and AlexaFluor 555), DyLight 649,Texas red, rhodamine B, and the like), radiolabels (e.g., ³H, ¹⁴C, ³⁵S,¹²⁵I, ¹²¹I, ¹¹²In, ⁹⁹mTc), other imaging agents such as microbubbles(for ultrasound imaging), ⁸F, ¹¹C, ¹⁵O, (for Positron emissiontomography), ⁹⁹mTC, ¹¹¹In (for Single photon emission tomography),gadolinium (Gd) chelate, terbium (Tb) chelate, dysprosium (Dy) chelate,europium (Eu) chelate, ytterbium (Yb) chelate or iron (for magneticresonance imaging), enzymes (e.g., horse radish peroxidase, alkalinephosphatase and others commonly used in an ELISA), and calorimetriclabels such as colloidal gold or colored glass or plastic (e.g.polystyrene, polypropylene, latex, and the like) beads. See alsoHandbook of Fluorescent Probes and Research Chemicals, 6^(th) Ed.,Molecular Probes, Inc., Eugene Oreg., which is incorporated herein byreference. In some embodiments, the functionalized Tymovirus virus orTymovirus VLPs are loaded with or conjugated to a contrast agent, suchas gadolinium (Gd) chelate, and a fluorescent dye, such as Cy5.5.

The label may be conjugated directly or indirectly via a linker to thedesired component of the Tymovirus virus or Tymovirus VLP according tomethods well known in the art. As indicated above, a wide variety oflabels may be used, with the choice of label depending on sensitivityrequired, ease of conjugation with the compound, stability requirements,available instrumentation, and disposal provisions.

Non-radioactive labels are often attached by indirect means. Generally,a ligand molecule (e.g., biotin) is covalently bound to the molecule.The ligand then binds to an anti-ligand (e.g., streptavidin) moleculewhich is either inherently detectable or covalently bound to a signalsystem, such as a detectable enzyme, a fluorescent compound, or achemiluminescent compound. A number of ligands and anti-ligands can beused. Where a ligand has a natural anti-ligand, for example, biotin,thyroxine, and cortisol, it can be used in conjunction with the labeled,naturally occurring anti-ligands.

The molecules can also be conjugated directly to signal generatingcompounds, e.g., by conjugation with an enzyme or fluorophore. Enzymesof interest as labels will primarily be hydrolases, particularlyphosphatases, esterases and glycosidases, or oxidoreductases,particularly peroxidases. Fluorescent compounds include compounds of theAlexa Fluor® series (Invitrogen™), fluorescein and its derivatives,rhodamine and its derivatives (e.g., rhodamine B), dansyl,umbelliferone, and the like Chemiluminescent compounds includeluciferin, and 2,3-dihydrophthalazinediones, e.g., luminol. For a reviewof various labeling or signal producing systems which may be used, see,U.S. Pat. No. 4,391,904, incorporated herein by reference.

In an exemplary embodiment, the surface exposed lysine residues of aTymovirus virus or Tymovirus VLP are conjugated to NHS-activated estersof the fluorophore sulfo-cyanine 5 succinimidyl ester (sulfo-Cy5).Similarly the thiol grops on the internal cysteine residues of theTymovirus virus or Tymovirus VLPs can be conjugated to Cy5.5-maleimide.

Means of detecting labels are well known to those of skill in the art.Thus, for example, where the label is a radioactive label, means fordetection include a scintillation counter or photographic film as inautoradiography. Where the label is a fluorescent label, it may bedetected by exciting the fluorochrome with the appropriate wavelength oflight and detecting the resulting fluorescence. The fluorescence may bedetected visually, by means of photographic film, by the use ofelectronic detectors such as charge coupled devices (CCDs) orphotomultipliers and the like. Similarly, enzymatic labels may bedetected by providing the appropriate substrates for the enzyme anddetecting the resulting reaction product. Finally simple calorimetriclabels may be detected simply by observing the color associated with thelabel.

In some embodiments, methods described herein can further include thestep of imaging the cancer tissue in the subject using an imaging devicewherein the cancer tissue is imaged subsequent to administering aneffective amount of the plurality of Tymovirus virus or Tymovirus VLPsincluding one or more imaging agents. Examples of imaging methodsinclude computed tomography, positive emission tomography, and magneticresonance imaging.

“Computed tomography (CT)” refers to a diagnostic imaging tool thatcomputes multiple x-ray cross sections to produce a cross-sectional viewof the vascular system, organs, bones, and tissues. “Positive emissionstomography (PET)” refers to a diagnostic imaging tool in which thepatient receives a radioactive isotopes by injection or ingestion whichthen computes multiple x-ray cross sections to produce a cross-sectionalview of the vascular system, organs, bones, and tissues to image theradioactive tracer. These radioactive isotopes are bound to compounds ordrugs that are injected into the body and enable study of the physiologyof normal and abnormal tissues. “Magnetic resonance imaging (MRI)”refers to a diagnostic imaging tool using magnetic fields and radiowavesto produce a cross-sectional view of the body including the vascularsystem, organs, bones, and tissues.

Therapeutic Agents

In certain embodiments, the Tymovirus virus or Tymovirus VLPs can beloaded with or conjugated to one or more therapeutic compounds, such asanti-cancer agents. In some embodiments, the Tymovirus virus orTymovirus VLPs can be loaded with or conjugated to one or moreanti-cancer agents, such as, but not limited to: acivicin, aclarubicin,acodazole hydrochloride, acronine, adozelesin, aldesleukin, altretamine,ambomycin, ametantrone acetate, aminoglutethimide, amsacrine,anastrozole, anthramycin, asparaginase, asperlin, azacitidine, azetepa,azotomycin, batimastat, benzodepa, bicalutamide, bisantrenehydrochloride, bisnafide dimesylate, bizelesin, bleomycin sulfate,brequinar sodium, bropirimine, busulfan, cactinomycin, calusterone,caracemide, carbetimer, carboplatin, carmustine, carubicinhydrochloride, carzelesin, cedefingol, chlorambucil, cirolemycin,cisplatin, cladribine, crisnatol mesylate, cyclophosphamide, cytarabine,dacarbazine, dactinomycin, daunorubicin hydrochloride, decarbazine,decitabine, dexormaplatin, dezaguanine, dezaguanine mesylate,diaziquone, docetaxel, doxorubicin, doxorubicin hydrochloride,droloxifene, droloxifene citrate, dromostanolone propionate, duazomycin,edatrexate, eflornithine hydrochloride, elsamitrucin, enloplatin,enpromate, epipropidine, epirubicin hydrochloride, erbulozole,esorubicin hydrochloride, estramustine, estramustine phosphate sodium,etanidazole, etoposide, etoposide phosphate, etoprine, fadrozolehydrochloride, fazarabine, fenretinide, floxuridine, fludarabinephosphate, fluorouracil, flurocitabine, fosquidone, fostriecin sodium,gemcitabine, gemcitabine hydrochloride, hydroxyurea, idarubicinhydrochloride, ifosfamide, ilmofosine, interleukin 2 (includingrecombinant interleukin 2, or rIL2), interferon alpha-2a, interferonalpha-2b, interferon alpha-n1, interferon alpha-n3, interferon beta-I a,interferon gamma-I b, iproplatin, irinotecan hydrochloride, lanreotideacetate, letrozole, leuprolide acetate, liarozole hydrochloride,lometrexol sodium, lomustine, losoxantrone hydrochloride, masoprocol,maytansine, mechlorethamine hydrochloride, megestrol acetate,melengestrol acetate, melphalan, menogaril, mercaptopurine,methotrexate, methotrexate sodium, metoprine, meturedepa, mitindomide,mitocarcin, mitocromin, mitogillin, mitomalcin, mitomycin, mitosper,mitotane, mitoxantrone hydrochloride, mycophenolic acid, nitrosoureas,nocodazole, nogalamycin, ormaplatin, oxisuran, paclitaxel, pegaspargase,peliomycin, pentamustine, peplomycin sulfate, perfosfamide, pipobroman,piposulfan, piroxantrone hydrochloride, plicamycin, plomestane, porfimersodium, porfiromycin, prednimustine, procarbazine hydrochloride,puromycin, puromycin hydrochloride, pyrazofurin, riboprine, rogletimide,safingol, safingol hydrochloride, semustine, simtrazene, sparfosatesodium, sparsomycin, spirogermanium hydrochloride, spiromustine,spiroplatin, streptonigrin, streptozocin, sulofenur, talisomycin,tecogalan sodium, tegafur, teloxantrone hydrochloride, temoporfin,teniposide, teroxirone, testolactone, thiamiprine, thioguanine,thiotepa, tiazofurin, tirapazamine, toremifene citrate, trestoloneacetate, triciribine phosphate, trimetrexate, trimetrexate glucuronate,triptorelin, tubulozole hydrochloride, uracil mustard, uredepa,vapreotide, verteporfin, vinblastine sulfate, vincristine sulfate,vindesine, vindesine sulfate, vinepidine sulfate, vinglycinate sulfate,vinleurosine sulfate, vinorelbine tartrate, vinrosidine sulfate,vinzolidine sulfate, vorozole, zeniplatin, zinostatin, zorubicinhydrochloride. Other anti-cancer drugs include, but are not limited to:20-epi-1,25 dihydroxyvitamin D3,5-ethynyluracil, abiraterone,aclarubicin, acylfulvene, adecypenol, adozelesin, aldesleukin, ALL-TKantagonists, altretamine, ambamustine, amidox, amifostine,aminolevulinic acid, amrubicin, amsacrine, anagrelide, anastrozole,andrographolide, angiogenesis inhibitors, antagonist D, antagonist G,antarelix, anti-dorsalizing morphogenetic protein-1, antiandrogens,antiestrogens, antineoplaston, aphidicolin glycinate, apoptosis genemodulators, apoptosis regulators, apurinic acid, ara-CDP-DL-PTBA,arginine deaminase, asulacrine, atamestane, atrimustine, axinastatin 1,axinastatin 2, axinastatin 3, azasetron, azatoxin, azatyrosine, baccatinIII derivatives, balanol, batimastat, BCR/ABL antagonists,benzochlorins, benzoylstaurosporine, beta lactam derivatives,beta-alethine, betaclamycin B, betulinic acid, bFGF inhibitor,bicalutamide, bisantrene, bisaziridinylspermine, bisnafide, bistrateneA, bizelesin, breflate, bropirimine, budotitane, buthionine sulfoximine,calcipotriol, calphostin C, camptothecin derivatives, canarypox IL-2,capecitabine, carboxamide-amino-triazole, carboxyamidotriazole, CaRestM3, CARN 700, cartilage derived inhibitor, carzelesin, casein kinaseinhibitors (ICOS), castanospermine, cecropin B, cetrorelix,chloroquinoxaline sulfonamide, cicaprost, cis-porphyrin, cladribine,clomifene analogues, clotrimazole, collismycin A, collismycin B,combretastatin A4, combretastatin analogue, conagenin, crambescidin 816,crisnatol, cryptophycin 8, cryptophycin A derivatives, curacin A,cyclopentanthraquinones, cycloplatam, cypemycin, cytarabine ocfosfate,cytolytic factor, cytostatin, dacliximab, decitabine, dehydrodidemnin B,deslorelin, dexamethasone, dexifosfamide, dexrazoxane, dexverapamil,diaziquone, didemnin B, didox, diethylnorspermine,dihydro-5-azacytidine, dihydrotaxol, dioxamycin, diphenyl spiromustine,docetaxel, docosanol, dolasetron, doxifluridine, droloxifene,dronabinol, duocarmycin SA, ebselen, ecomustine, edelfosine,edrecolomab, eflomithine, elemene, emitefur, epirubicin, epristeride,estramustine analogue, estrogen agonists, estrogen antagonists,etanidazole, etoposide phosphate, exemestane, fadrozole, fazarabine,fenretinide, filgrastim, finasteride, flavopiridol, flezelastine,fluasterone, fludarabine, fluorodaunorunicin hydrochloride, forfenimex,formestane, fostriecin, fotemustine, gadolinium texaphyrin, galliumnitrate, galocitabine, ganirelix, gelatinase inhibitors, gemcitabine,glutathione inhibitors, hepsulfam, heregulin, hexamethylenebisacetamide, hypericin, ibandronic acid, idarubicin, idoxifene,idramantone, ilmofosine, ilomastat, imidazoacridones, imiquimod,immunostimulant peptides, insulin-like growth factor-1 receptorinhibitor, interferon agonists, interferons, interleukins, iobenguane,iododoxorubicin, ipomeanol, iroplact, irsogladine, isobengazole,isohomohalicondrin B, itasetron, jasplakinolide, kahalalide F,lamellarin-N triacetate, lanreotide, leinamycin, lenograstim, lentinansulfate, leptolstatin, letrozole, leukemia inhibiting factor, leukocytealpha interferon, leuprolide+estrogen+progesterone, leuprorelin,levamisole, liarozole, linear polyamine analogue, lipophilicdisaccharide peptide, lipophilic platinum compounds, lissoclinamide 7,lobaplatin, lombricine, lometrexol, lonidamine, losoxantrone,lovastatin, loxoribine, lurtotecan, lutetium texaphyrin, lysofylline,lytic peptides, maitansine, mannostatin A, marimastat, masoprocol,maspin, matrilysin inhibitors, matrix metalloproteinase inhibitors,menogaril, merbarone, meterelin, methioninase, metoclopramide, MIFinhibitor, mifepristone, miltefosine, mirimostim, mismatched doublestranded RNA, mitoguazone, mitolactol, mitomycin analogues, mitonafide,mitotoxin fibroblast growth factor-saporin, mitoxantrone, mofarotene,molgramostim, monoclonal antibody, human chorionic gonadotrophin,monophosphoryl lipid A+myobacterium cell wall sk, mopidamol, multipledrug resistance gene inhibitor, multiple tumor suppressor 1 basedtherapy, mustard anticancer agent, mycaperoxide B, mycobacterial cellwall extract, myriaporone, N-acetyldinaline, N-substituted benzamides,nafarelin, nagrestip, naloxone+pentazocine, napavin, naphterpin,nartograstim, nedaplatin, nemorubicin, neridronic acid, neutralendopeptidase, nilutamide, nisamycin, nitric oxide modulators, nitroxideantioxidant, nitrullyn, O6-benzylguanine, octreotide, okicenone,oligonucleotides, onapristone, ondansetron, ondansetron, oracin, oralcytokine inducer, ormaplatin, osaterone, oxaliplatin, oxaunomycin,paclitaxel, paclitaxel analogues, paclitaxel derivatives, palauamine,palmitoylrhizoxin, pamidronic acid, panaxytriol, panomifene, parabactin,pazelliptine, pegaspargase, peldesine, pentosan polysulfate sodium,pentostatin, pentrozole, perflubron, perfosfamide, perillyl alcohol,phenazinomycin, phenylacetate, phosphatase inhibitors, picibanil,pilocarpine hydrochloride, pirarubicin, piritrexim, placetin A, placetinB, plasminogen activator inhibitor, platinum complex, platinumcompounds, platinum-triamine complex, porfimer sodium, porfiromycin,prednisone, propyl bis-acridone, prostaglandin J2, proteasomeinhibitors, protein A-based immune modulator, protein kinase Cinhibitor, protein kinase C inhibitors, microalgal, protein tyrosinephosphatase inhibitors, purine nucleoside phosphorylase inhibitors,purpurins, pyrazoloacridine, pyridoxylated hemoglobin polyoxyethyleneconjugate, raf antagonists, raltitrexed, ramosetron, ras farnesylprotein transferase inhibitors, ras inhibitors, ras-GAP inhibitor,retelliptine demethylated, rhenium Re 186 etidronate, rhizoxin,ribozymes, R11 retinamide, rogletimide, rohitukine, romurtide,roquinimex, rubiginone B 1, ruboxyl, safingol, saintopin, SarCNU,sarcophytol A, sargramostim, Sdi 1 mimetics, semustine, senescencederived inhibitor 1, sense oligonucleotides, signal transductioninhibitors, signal transduction modulators, single chain antigen bindingprotein, sizofiran, sobuzoxane, sodium borocaptate, sodiumphenylacetate, solverol, somatomedin binding protein, sonermin,sparfosic acid, spicamycin D, spiromustine, splenopentin, spongistatin1, squalamine, stem cell inhibitor, stem-cell division inhibitors,stipiamide, stromelysin inhibitors, sulfinosine, superactive vasoactiveintestinal peptide antagonist, suradista, suramin, swainsonine,synthetic glycosaminoglycans, tallimustine, tamoxifen methiodide,tauromustine, taxol, tazarotene, tecogalan sodium, tegafur,tellurapyrylium, telomerase inhibitors, temoporfin, temozolomide,teniposide, tetrachlorodecaoxide, tetrazomine, thaliblastine,thalidomide, thiocoraline, thioguanine, thrombopoietin, thrombopoietinmimetic, thymalfasin, thymopoietin receptor agonist, thymotrinan,thyroid stimulating hormone, tin ethyl etiopurpurin, tirapazamine,titanocene bichloride, topsentin, toremifene, totipotent stem cellfactor, translation inhibitors, tretinoin, triacetyluridine,triciribine, trimetrexate, triptorelin, tropisetron, turosteride,tyrosine kinase inhibitors, tyrphostins, UBC inhibitors, ubenimex,urogenital sinus-derived growth inhibitory factor, urokinase receptorantagonists, vapreotide, variolin B, vector system, erythrocyte genetherapy, velaresol, veramine, verdins, verteporfin, vinorelbine,vinxaltine, vitaxin, vorozole, zanoterone, zeniplatin, zilascorb, andzinostatin stimalamer.

In some embodiments, the therapeutic compounds include cytotoxiccompounds. It has been shown that the cytotoxicity of a therapeuticagent is not significantly affected by inclusion in a Tymovirus virus orTymovirus VLP described herein. Thus, in certain embodiments, one ormore cytotoxic compounds included in a Tymovirus virus or Tymovirus VLPretain their cytotoxic activity. The inclusion of a therapeutic agent ina Tymovirus virus or Tymovirus VLP that is preferentially internalizedby cancer cells allows for the delivery of highly potent therapeuticagents to a subject's cancer cells while overcoming the dose-limitingtoxicity of the drug towards healthy cells.

Cytotoxic compounds for use in a method or composition described hereininclude compounds that inhibit cell growth or promote cell death whenproximate to or absorbed by a cell. Suitable cytotoxic compounds in thisregard include radioactive agents or isotopes (radionuclides),chemotoxic agents such as differentiation inducers, inhibitors and smallchemotoxic drugs, toxin proteins and derivatives thereof, as well asnucleotide sequences (or their antisense sequence). Therefore, thecytotoxic compound can be, by way of non-limiting example, an antitumoragent, a photoactivated toxin or a radioactive agent.

Preferred radionuclides for use as cytotoxic compounds are radionuclideswhich are suitable for pharmacological administration. Suchradionuclides include ¹²³I, ¹²⁵I, ¹³¹I, ⁹⁰Y, ²¹¹At, ⁶⁷Cu, ¹⁸⁶Re, ¹⁸⁸Re,²¹²Pb, and ²¹²Bi. Iodine and astatine isotopes are more preferredradionuclides for use in the therapeutic compositions of the presentinvention, as a large body of literature has been accumulated regardingtheir use. ¹³¹I is particularly preferred, as are other β-radiationemitting nuclides, which have an effective range of several millimeters.¹²³I, ¹²⁵I, ¹³¹I, or ²¹¹At can be conjugated to Tymovirus virus orTymovirus VLPs for use in the compositions and methods utilizing any ofseveral known conjugation reagents, including Iodogen, N-succinimidyl3-[²¹¹At]astatobenzoate, N-succinimidyl 3-[¹³¹I]iodobenzoate (SIB), and,N-succinimidyl 5-[¹³¹I]iodo-3-pyridinecarboxylate (SIPC). Any iodineisotope can be utilized in the recited iodo-reagents. Otherradionuclides can be conjugated to the Tymovirus virus or Tymovirus VLPsby suitable chelation agents known to those of skill in the nuclearmedicine arts.

In certain embodiments, cytotoxic compounds include small-molecule drugssuch as doxorubicin, mitoxantrone, methotrexate, and pyrimidine andpurine analogs, referred to herein as antitumor agents. Preferredchemotoxin differentiation inducers include phorbol esters and butyricacid. Antitumor agents can be directly conjugated to the Tymovirus virusor Tymovirus VLPs via a chemical linker, or can be encapsulated in acarrier, which is in turn coupled to the Tymovirus virus or TymovirusVLPs. In certain embodiments, where encapsulation is not preferred orfeasible, cytotoxic compounds or imaging agents can be directly infusedinto the Tymovirus virus or Tymovirus VLPs using a non covalent infusionprotocol. For example, Tymovirus virus or Tymovirus VLPs can beincubated with a molar excess of about 500, about 2000, about 5000, orabout 10000 cargo molecules (e.g., a dye or cytotoxic agent) perparticle overnight at room temperature in the dark and then purified toremove excess reagents. In certain embodiments, the cytotoxicity of thefree cytotoxic agent is not significantly affected by encapsulation bythe Tymovirus virus or Tymovirus VLPs.

Preferred toxin proteins for use as cytotoxic compounds include ricin,abrin, diphtheria toxin, cholera toxin, gelonin, Pseudomonas exotoxin,Shigella toxin, pokeweed antiviral protein, and other toxin proteinsknown in the medicinal biochemistry arts. As these toxin agents canelicit undesirable immune responses in the patient, especially ifinjected intravascularly, it is preferred that they be encapsulated in acarrier for coupling to the Tymovirus virus or Tymovirus VLPs.

In certain embodiments of the invention, the Tymovirus virus orTymovirus VLPs can be loaded with or conjugated to one or morephotodynamic therapeutic photosensitizer (PDT sensitizer) compounds.Photodynamic therapeutic photosensitizer compounds are compounds thatare excited by an appropriate light source to produce radicals and/orreactive oxygen species. Typically, when a sufficient amount ofphotosensitizer appears in diseased tissue (e.g., tumor tissue), thephotosensitizer can be activated by exposure to light for a specifiedperiod. The light dose supplies sufficient energy to stimulate thephotosensitizer, but not enough to damage neighboring healthy tissue.The radicals or reactive oxygen produced following photosensitizerexcitation kill the target cells (e.g., cancer cells). In someembodiments, the targeted tissue can be locally illuminated. Forexample, light can be delivered to a photosensitizer via an argon orcopper pumped dye laser coupled to an optical fiber, a double laserconsisting of KTP (potassium titanyl phosphate)/YAG (yttrium aluminumgarnet) medium, LED (light emitting diode), or a solid state laser.

PDT sensitizers for use in a method and/or composition described hereincan include a first generation photosensitizer (e.g., hematoporphyrinderivatives (HpDs) such as Photofrin (porfimer sodium), Photogem,Photosan-3 and the like). In some embodiments, PDT sensitizers caninclude second and third generation photosensitizers such asporphyrinoid derivatives and precursors. Porphyrinoid derivatives andprecursors can include porphyrins and mettaloporphrins (e.g.,meta-tetra(hydroxyphenyl)porphyrin (m-THPP),5,10,15,20-tetrakis(4-sulfanatophenyl)-21H,23H-porphyrin (TPPS₄), andprecursors to endogenous protoporphyrin IX (PpIX): 1,5-aminolevulinicacid (ALA), methyl aminolevulinate (MAL), hexaminolevulinate (HAL)),chlorins (e.g., benzoporphyrin derivative monoacid ring A (BPD-MA),meta-tetra(hydroxyphenyl)chlorin (m-THPC), N-aspartyl chlorin e6 (NPe6),and tin ethyl etiopurpurin (SnET2)), pheophorbides (e.g.,2-(1-hexyloxyethyl)-2-devinyl pyropheophorbide (HPPH)),bacteriopheophorbides (e.g., bacteriochlorphyll a, WST09 and WST11),Texaphyrins (e.g., motexafin lutetium (Lu-Tex)), and phthalocyanines(PCs) (e.g., aluminum phthalocyanine tetrasulfonate (AlPcS4) and siliconphthalocyanine (Pc4)). In some embodiments, the PDT sensitizer caninclude cationic zinc ethynylphenyl porphyrin.

Although porphyrinoid structures comprise a majority ofphotosensitizers, several non-porphyrin chromogens exhibit photodynamicactivity. These compounds include anthraquinones, phenothiazines,xanthenes, cyanines, and curcuminoids.

Due in part to their preferential uptake/internalization by cancer cellsover non-cancerous cells, functionalized Tymovirus virus or TymovirusVLPs loaded with or conjugated one or more therapeutic agents (e.g.,cytotoxic compounds or PDT agents) can be used to treat a variety ofdifferent types of cancer. “Cancer” or “malignancy” are used assynonymous terms and refer to any of a number of diseases that arecharacterized by uncontrolled, abnormal proliferation of cells, theability of affected cells to spread locally or through the bloodstreamand lymphatic system to other parts of the body (i.e., metastasize) aswell as any of a number of characteristic structural and/or molecularfeatures.

A “cancer cell” refers to a cell undergoing early, intermediate oradvanced stages of multi-step neoplastic progression. The features ofearly, intermediate and advanced stages of neoplastic progression havebeen described using microscopy. Cancer cells at each of the threestages of neoplastic progression generally have abnormal karyotypes,including translocations, inversion, deletions, isochromosomes,monosomies, and extra chromosomes. Cancer cells include “hyperplasticcells,” that is, cells in the early stages of malignant progression,“dysplastic cells,” that is, cells in the intermediate stages ofneoplastic progression, and “neoplastic cells,” that is, cells in theadvanced stages of neoplastic progression.

The cancers treated by a method described herein can include thefollowing: leukemias, such as but not limited to, acute leukemia, acutelymphocytic leukemia, acute myelocytic leukemias, such as, myeloblastic,promyelocytic, myelomonocytic, monocytic, and erythroleukemia leukemiasand myelodysplastic syndrome; chronic leukemias, such as but not limitedto, chronic myelocytic (granulocytic) leukemia, chronic lymphocyticleukemia, hairy cell leukemia; polycythemia vera; lymphomas such as butnot limited to Hodgkin's disease, non-Hodgkin's disease; multiplemyelomas such as but not limited to smoldering multiple myeloma,nonsecretory myeloma, osteosclerotic myeloma, plasma cell leukemia,solitary plasmacytoma and extramedullary plasmacytoma; Waldenstrom'smacroglobulinemia; monoclonal gammopathy of undetermined significance;benign monoclonal gammopathy; heavy chain disease; bone and connectivetissue sarcomas such as but not limited to bone sarcoma, osteosarcoma,chondrosarcoma, Ewing's sarcoma, malignant giant cell tumor,fibrosarcoma of bone, chordoma, periosteal sarcoma, soft-tissuesarcomas, angiosarcoma (hemangiosarcoma), fibrosarcoma, Kaposi'ssarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, neurilemmoma,rhabdomyosarcoma, synovial sarcoma; brain tumors such as but not limitedto, glioma, astrocytoma, glioblastoma, brain stem glioma, ependymoma,oligodendroglioma, nonglial tumor, acoustic neurinoma,craniopharyngioma, medulloblastoma, meningioma, pineocytoma,pineoblastoma, primary brain lymphoma; breast cancer including but notlimited to ductal carcinoma, adenocarcinoma, lobular (small cell)carcinoma, intraductal carcinoma, medullary breast cancer, mucinousbreast cancer, tubular breast cancer, papillary breast cancer, Paget'sdisease, and inflammatory breast cancer; adrenal cancer such as but notlimited to pheochromocytoma and adrenocortical carcinoma; thyroid cancersuch as but not limited to papillary or follicular thyroid cancer,medullary thyroid cancer and anaplastic thyroid cancer; pancreaticcancer such as but not limited to, insulinoma, gastrinoma, glucagonoma,vipoma, somatostatin-secreting tumor, and carcinoid or islet cell tumor;pituitary cancers such as but limited to Cushing's disease,prolactin-secreting tumor, acromegaly, and diabetes insipius; eyecancers such as but not limited to ocular melanoma such as irismelanoma, choroidal melanoma, and cilliary body melanoma, andretinoblastoma; vaginal cancers such as squamous cell carcinoma,adenocarcinoma, and melanoma; vulvar cancer such as squamous cellcarcinoma, melanoma, adenocarcinoma, basal cell carcinoma, sarcoma, andPaget's disease; cervical cancers such as but not limited to, squamouscell carcinoma, and adenocarcinoma; uterine cancers such as but notlimited to endometrial carcinoma and uterine sarcoma; ovarian cancerssuch as but not limited to, ovarian epithelial carcinoma, borderlinetumor, germ cell tumor, fallopian tube cancer, and stromal tumor;esophageal cancers such as but not limited to, squamous cancer,adenocarcinoma, adenoid cystic carcinoma, mucoepidermoid carcinoma,adenosquamous carcinoma, sarcoma, melanoma, plasmacytoma, verrucouscarcinoma, and oat cell (small cell) carcinoma; stomach cancers such asbut not limited to, adenocarcinoma, fungating (polypoid), ulcerating,superficial spreading, diffusely spreading, malignant lymphoma,liposarcoma, fibrosarcoma, and carcinosarcoma; colon cancers; rectalcancers; liver cancers such as but not limited to hepatocellularcarcinoma and hepatoblastoma; gallbladder cancers such asadenocarcinoma; cholangiocarcinomas such as but not limited topapillary, nodular, and diffuse; lung cancers such as non-small celllung cancer, squamous cell carcinoma (epidermoid carcinoma),adenocarcinoma, large-cell carcinoma and small-cell lung cancer;testicular cancers such as but not limited to germinal tumor, seminoma,anaplastic, classic (typical), spermatocytic, nonseminoma, embryonalcarcinoma, teratoma carcinoma, choriocarcinoma (yolk-sac tumor),prostate cancers such as but not limited to, prostatic intraepithelialneoplasia, adenocarcinoma, leiomyosarcoma, and rhabdomyosarcoma; penalcancers; oral cancers such as but not limited to squamous cellcarcinoma; basal cancers; salivary gland cancers such as but not limitedto adenocarcinoma, mucoepidermoid carcinoma, and adenoidcysticcarcinoma; pharynx cancers such as but not limited to squamous cellcancer, and verrucous; skin cancers such as but not limited to, basalcell carcinoma, squamous cell carcinoma and melanoma, superficialspreading melanoma, nodular melanoma, lentigo malignant melanoma, acrallentiginous melanoma; kidney cancers such as but not limited to renalcell carcinoma, adenocarcinoma, hypemephroma, fibrosarcoma, transitionalcell cancer (renal pelvis and/or uterer); Wilms' tumor; bladder cancerssuch as but not limited to transitional cell carcinoma, squamous cellcancer, adenocarcinoma, carcinosarcoma. In addition, cancers includemyxosarcoma, osteogenic sarcoma, endotheliosarcoma,lymphangioendotheliosarcoma, mesothelioma, synovioma, hemangioblastoma,epithelial carcinoma, cystadenocarcinoma, bronchogenic carcinoma, sweatgland carcinoma, sebaceous gland carcinoma, papillary carcinoma andpapillary adenocarcinomas (for a review of such disorders, see Fishmanet al., 1985, Medicine, 2d Ed., J. B. Lippincott Co., Philadelphia andMurphy et al., 1997, Informed Decisions: The Complete Book of CancerDiagnosis, Treatment, and Recovery, Viking Penguin, Penguin BooksU.S.A., Inc., United States of America).

In certain embodiments, the functionalized Tymovirus virus or TymovirusVLPs are used to treat and/or image cancer tissue selected from thegroup consisting of ovarian, breast and prostate cancer.

Targeting Agents

In some embodiments, the Tymovirus virus or Tymovirus VLPs canadditionally or optionally be loaded with or conjugated to one or moretargeting agents that are capable of targeting and/or adhering theTymovirus virus or Tymovirus VLPs to a cell or tissue of interest. Insome embodiments, the use of targeting agents can enhance the deliveryof the virus or VLPs to a targeted site and/or the preferentialinternalization of Tymovirus virus or Tymovirus VLPs exhibited by cancercells over non-cancerous cells. The targeting agent can comprise anymolecule, or complex of molecules, which is/are capable of interactingwith an intracellular, cell surface, or extracellular biomarker of thecell. The biomarker can include, for example, a cellular protease, akinase, a protein, a cell surface receptor, a lipid, and/or fatty acid.Other examples of biomarkers that the targeting agent can interact withinclude molecules associated with a particular disease. For example, thebiomarkers can include cell surface receptors implicated in cancerdevelopment, such as epidermal growth factor receptor and transferrinreceptor, or cancer metastasis, such as α_(v)β₃ or α₂β₁ integrin. Thetargeting moieties can interact with the biomarkers through, forexample, non-covalent binding, covalent binding, hydrogen binding, vander Waals forces, ionic bonds, hydrophobic interactions, electrostaticinteraction, and/or combinations thereof.

The targeting agents can include, but are not limited to, syntheticcompounds, natural compounds or products, macromolecular entities,bioengineered molecules (e.g., polypeptides, lipids, polynucleotides,antibodies, antibody fragments), and small entities (e.g., smallmolecules, neurotransmitters, substrates, ligands, hormones andelemental compounds).

In one example, the targeting agent can include an antibody, such as amonoclonal antibody, a polyclonal antibody, or a humanized antibody. Theantibody can include Fv fragments, single chain Fv (scFv) fragments,Fab′ fragments, F(ab′)2 fragments, single domain antibodies, camelizedantibodies and other antibody fragments. The antibody can also includemultivalent versions of the foregoing antibodies or fragments thereofincluding monospecific or bispecific antibodies, such as disulfidestabilized Fv fragments, scFv tandems ((scFv)₂ fragments), diabodies,tribodies or tetrabodies, which typically are covalently linked orotherwise stabilized (i.e., leucine zipper or helix stabilized) scFvfragments; and receptor molecules, which naturally interact with adesired target molecule.

Preparation of antibodies can be accomplished by any number of methodsfor generating antibodies. These methods typically include the step ofimmunization of animals, such as mice or rabbits, with a desiredimmunogen (e.g., a desired target molecule or fragment thereof). Oncethe mammals have been immunized, and boosted one or more times with thedesired immunogen(s), antibody-producing hybridomas may be prepared andscreened according to well known methods. See, for example, Kuby, Janis,Immunology, Third Edition, pp. 131-139, W.H. Freeman & Co. (1997), for ageneral overview of monoclonal antibody production, that portion ofwhich is incorporated herein by reference.

In vitro methods that combine antibody recognition and phage displaytechniques can also be used to allow one to amplify and selectantibodies with very specific binding capabilities. See, for example,Holt, L. J. et al., “The Use of Recombinant Antibodies in Proteomics,”Current Opinion in Biotechnology, 2000, 11:445-449, incorporated hereinby reference. These methods typically are much less cumbersome thanpreparation of hybridomas by traditional monoclonal antibody preparationmethods.

In some embodiments, phage display technology may be used to generate atargeting agent specific for a desired target molecule. An immuneresponse to a selected immunogen is elicited in an animal (such as amouse, rabbit, goat or other animal) and the response is boosted toexpand the immunogen-specific B-cell population. Messenger RNA isisolated from those B-cells, or optionally a monoclonal or polyclonalhybridoma population. The mRNA is reverse-transcribed by known methodsusing either a poly-A primer or murine immunoglobulin-specificprimer(s), typically specific to sequences adjacent to the desired V_(H)and V_(L) chains, to yield cDNA. The desired V_(H) and V_(L) chains areamplified by polymerase chain reaction (PCR) typically using V_(H) andV_(L) specific primer sets, and are ligated together, separated by alinker. V_(H) and V_(L) specific primer sets are commercially available,for instance from Stratagene, Inc. of La Jolla, Calif. AssembledV_(H)-linker-V_(L) product (encoding a scFv fragment) is selected forand amplified by PCR. Restriction sites are introduced into the ends ofthe V_(H)-linker-V_(L) product by PCR with primers including restrictionsites and the scFv fragment is inserted into a suitable expressionvector (typically a plasmid) for phage display. Other fragments, such asa Fab′ fragment, may be cloned into phage display vectors for surfaceexpression on phage particles. The phage may be any phage, such aslambda, but typically is a filamentous phage, such as Fd and M13,typically M13.

In phage display vectors, the V_(H)-linker-V_(L) sequence is cloned intoa phage surface protein (for M13, the surface proteins g3p (pIII) org8p, most typically g3p). Phage display systems also include phagemidsystems, which are based on a phagemid plasmid vector containing thephage surface protein genes (for example, g3p and g8p of M13) and thephage origin of replication. To produce phage particles, cellscontaining the phagemid are rescued with helper phage providing theremaining proteins needed for the generation of phage. Only the phagemidvector is packaged in the resulting phage particles because replicationof the phagemid is grossly favored over replication of the helper phageDNA. Phagemid packaging systems for production of antibodies arecommercially available. One example of a commercially available phagemidpackaging system that also permits production of soluble ScFv fragmentsin bacterial cells is the Recombinant Phage Antibody System (RPAS),commercially available from Amersham Pharmacia Biotech, Inc. ofPiscataway, N.J. and the pSKAN Phagemid Display System, commerciallyavailable from MoBiTec, LLC of Marco Island, Fla. Phage display systems,their construction, and screening methods are described in detail in,among others, U.S. Pat. Nos. 5,702,892, 5,750,373, 5,821,047 and6,127,132, each of which is incorporated herein by reference in theirentirety.

The targeting agent need not originate from a biological source. Thetargeting agent may, for example, be screened from a combinatoriallibrary of synthetic peptides. One such method is described in U.S. Pat.No. 5,948,635, incorporated herein by reference, which described theproduction of phagemid libraries having random amino acid insertions inthe pIII gene of M13. These phage may be clonally amplified by affinityselection as described above.

The immunogens used to prepare targeting moieties having a desiredspecificity will generally be the target molecule, or a fragment orderivative thereof. Such immunogens may be isolated from a source wherethey are naturally occurring or may be synthesized using methods knownin the art. For example, peptide chains may be synthesized by1-ethyl-3-[dimethylaminoproply]carbodiimide (EDC)-catalyzed condensationof amine and carboxyl groups. In certain embodiments, the immunogen maybe linked to a carrier bead or protein. For example, the carrier may bea functionalized bead such as SASRIN resin commercially available fromBachem, King of Prussia, Pa. or a protein such as keyhole limpethemocyanin (KLH) or bovine serum albumin (BSA). The immunogen may beattached directly to the carrier or may be associated with the carriervia a linker, such as a non-immunogenic synthetic linker (for example, apolyethylene glycol (PEG) residue, amino caproic acid or derivativesthereof) or a random, or semi-random polypeptide.

In certain embodiments, it may be desirable to mutate the binding regionof the polypeptide targeting agent and select for a targeting agent withsuperior binding characteristics as compared to the un-mutated targetingagent. This may be accomplished by any standard mutagenesis technique,such as by PCR with Taq polymerase under conditions that cause errors.In such a case, the PCR primers could be used to amplify scFv-encodingsequences of phagemid plasmids under conditions that would causemutations. The PCR product may then be cloned into a phagemid vector andscreened for the desired specificity, as described above.

In other embodiments, the targeting agents may be modified to make themmore resistant to cleavage by proteases. For example, the stability oftargeting agent comprising a polypeptide may be increased bysubstituting one or more of the naturally occurring amino acids in the(L) configuration with D-amino acids. In various embodiments, at least1%, 5%, 10%, 20%, 50%, 80%, 90% or 100% of the amino acid residues oftargeting agent may be of the D configuration. The switch from L to Damino acids neutralizes the digestion capabilities of many of theubiquitous peptidases found in the digestive tract. Alternatively,enhanced stability of a targeting agent comprising a peptide bond may beachieved by the introduction of modifications of the traditional peptidelinkages. For example, the introduction of a cyclic ring within thepolypeptide backbone may confer enhanced stability in order tocircumvent the effect of many proteolytic enzymes known to digestpolypeptides in the stomach or other digestive organs and in serum. Instill other embodiments, enhanced stability of a targeting moiety may beachieved by intercalating one or more dextrorotatory amino acids (suchas, dextrorotatory phenylalanine or dextrorotatory tryptophan) betweenthe amino acids of targeting moiety. In exemplary embodiments, suchmodifications increase the protease resistance of a targeting agentwithout affecting the activity or specificity of the interaction with adesired target molecule.

In certain embodiments, a targeting agent as described herein maycomprise a homing peptide, which selectively directs the nanoparticle toa targeted cell. Homing peptides for a targeted cell can be identifiedusing various methods well known in the art. Many laboratories haveidentified the homing peptides that are selective for cells of thevasculature of brain, kidney, lung, skin, pancreas, intestine, uterus,adrenal gland, retina, muscle, prostate, or tumors. See, for example,Samoylova et al., 1999, Muscle Nerve, 22:460; Pasqualini et al., 1996Nature, 380:364; Koivunen et al., 1995, Biotechnology, 13:265;Pasqualini et al., 1995, J. Cell Biol., 130:1189; Pasqualini et al.,1996, Mole. Psych., 1:421, 423; Rajotte et al., 1998, J. Clin. Invest.,102:430; Rajotte et al., 1999, J. Biol. Chem., 274:11593. See, also,U.S. Pat. Nos. 5,622,6999; 6,068,829; 6,174,687; 6,180,084; 6,232,287;6,296,832; 6,303,573; and 6,306,365.

Phage display technology provides a means for expressing a diversepopulation of random or selectively randomized peptides. Various methodsof phage display and methods for producing diverse populations ofpeptides are well known in the art. For example, methods for preparingdiverse populations of binding domains on the surface of a phage havebeen described in U.S. Pat. No. 5,223,409. In particular, phage vectorsuseful for producing a phage display library as well as methods forselecting potential binding domains and producing randomly orselectively mutated binding domains are also provided in U.S. Pat. No.5,223,409. Similarly, methods of producing phage peptide displaylibraries, including vectors and methods of diversifying the populationof peptides that are expressed, are also described in Smith et al.,1993, Meth. Enzymol., 217:228-257, Scott et al., Science, 249:386-390,and two PCT publications WO 91/07141 and WO 91/07149. Phage displaytechnology can be particularly powerful when used, for example, with acodon based mutagenesis method, which can be used to produce randompeptides or randomly or desirably biased peptides (see, e.g., U.S. Pat.No. 5,264,563). These or other well-known methods can be used to producea phage display library, which can be subjected to the in vivo phagedisplay method in order to identify a peptide that homes to one or a fewselected tissues.

In vitro screening of phage libraries has previously been used toidentify peptides that bind to antibodies or cell surface receptors(see, e.g., Smith, et al., 1993, Meth. Enzymol., 217:228-257). Forexample, in vitro screening of phage peptide display libraries has beenused to identify novel peptides that specifically bind to integrinadhesion receptors (see, e.g., Koivunen et al., 1994, J. Cell Biol.124:373-380), and to the human urokinase receptor (Goodson, et al.,1994, Proc. Natl. Acad. Sci., USA 91:7129-7133).

In certain embodiments, the targeting agent may comprise a receptormolecule, including, for example, receptors, which naturally recognize aspecific desired molecule of a target cell. Such receptor moleculesinclude receptors that have been modified to increase their specificityof interaction with a target molecule, receptors that have been modifiedto interact with a desired target molecule not naturally recognized bythe receptor, and fragments of such receptors (see, e.g., Skerra, 2000,J. Molecular Recognition, 13:167-187). A preferred receptor is achmokine receptor. Exemplary chemokine receptors have been described in,for example, Lapidot et al, 2002, Exp Hematol, 30:973-81 and Onuffer etal, 2002, Trends Pharmacol Sci, 23:459-67.

In some embodiments, the targeting agent can include cyclo(ARG-GLY-ASP-D-Phe-Cys) or (cRGDfC), which is a ligand for vasculartargeting and metastasis. In some embodiments, a detergent compatiblecan be used to quantify the number of peptides per FeMSN particles.

In other embodiments, the targeting agent can be targeting peptidecomprising an EGF peptide. The EGF peptide may comprise the amino acidsequence YHWYGYTPQNVI-amide. The peptide may be synthesized by anymethod known in the art. For example, the EGF peptide may be synthesizedmanually using Fmoc protected amino acids (Peptides International,Louisville, Ky.) on rink-amide CLEAR resin (Peptides International,Louisville, Ky., 100-200 mesh size, 0.4 milliequivalents/gram).

In still other embodiments, the targeting agent may comprise a ligandmolecule, including, for example, ligands which naturally recognize aspecific desired receptor of a target cell, such as a Transferrin (Tf)ligand. Such ligand molecules include ligands that have been modified toincrease their specificity of interaction with a target receptor,ligands that have been modified to interact with a desired receptor notnaturally recognized by the ligand, and fragments of such ligands.

In other embodiments, the targeting agent may comprise an aptamer.Aptamers are oligonucleotides that are selected to bind specifically toa desired molecular structure of the target cell. Aptamers typically arethe products of an affinity selection process similar to the affinityselection of phage display (also known as in vitro molecular evolution).The process involves performing several tandem iterations of affinityseparation, e.g., using a solid support to which the diseased immunogenis bound, followed by polymerase chain reaction (PCR) to amplify nucleicacids that bound to the immunogens. Each round of affinity separationthus enriches the nucleic acid population for molecules thatsuccessfully bind the desired immunogen. In this manner, a random poolof nucleic acids may be “educated” to yield aptamers that specificallybind target molecules. Aptamers typically are RNA, but may be DNA oranalogs or derivatives thereof, such as, without limitation, peptidenucleic acids (PNAs) and phosphorothioate nucleic acids.

In yet other embodiments, the targeting agent may be a peptidomimetic.By employing, for example, scanning mutagenesis to map the amino acidresidues of a protein, which is involved in binding other proteins,peptidomimetic compounds can be generated which mimic those residueswhich facilitate the interaction. Such mimetics may then be used as atargeting agent to deliver the composition to a target cell. Forinstance, non-hydrolyzable peptide analogs of such resides can begenerated using benzodiazepine (e.g., see Freidinger et al. in Peptides:Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden,Netherlands, 1988), azepine (e.g., see Huffman et al. in Peptides:Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden,Netherlands, 1988), substituted gamma lactam rings (Garvey et al. inPeptides: Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher:Leiden, Netherlands, 1988), keto-methylene pseudopeptides (Ewenson etal., 1986, J Med Chem 29:295; and Ewenson et al., in Peptides: Structureand Function (Proceedings of the 9th American Peptide Symposium) PierceChemical Co. Rockland, Ill., 1985), b-turn dipeptide cores (Nagai etal., 1985, Tetrahedron Lett 26:647; and Sato et al., 1986, J Chem SocPerkin Trans 1:1231), and β-aminoalcohols (Gordon et al., 1985, BiochemBiophys Res Cummun 126:419; and Dann et al., 1986, Biochem Biophys ResCommun 134:71).

The targeting agent may be attached directly to the Tymovirus virus orTymovirus VLP. In an exemplary embodiment, a targeting agent may beconjugated onto a Tymovirus virus or Tymovirus VLP via maleimidechemistry. In some embodiments, the targeting agent may be associatedwith or coupled to the nanoparticles using a linker. The linker can beof any suitable length and contain any suitable number of atoms and/orsubunits. The linker can include one or combination of chemical and/orbiological moieties. Examples of chemical moieties can include alkylgroups, methylene carbon chains, ether, polyether, alkyl amide linkers,alkenyl chains, alkynyl chains, disulfide groups, and polymers, such aspoly(ethylene glycol) (PEG), functionalized PEG, PEG-chelant polymers,dendritic polymers, and combinations thereof. Examples of biologicalmoieties can include peptides, modified peptides, streptavidin-biotin oravidin-biotin, polyaminoacids (e.g., polylysine), polysaccharides,glycosaminoglycans, oligonucleotides, phospholipid derivatives, andcombinations thereof.

In some embodiments, the Tymovirus virus or Tymovirus VLPs can includemultiple types of targeting moieties and the spacing and location of thetargeting moieties on each nanoparticle can be controlled to facilitatedelivery, targeting, and/or therapeutic efficacy of the nanoparticlecargo agent(s).

Immune Response to Virus-Like Particles

In some embodiments, administering a plurality of Tymovirus virus orTymovirus VLPs to a subject can generate an immune response. An “immuneresponse” refers to the concerted action of lymphocytes, antigenpresenting cells, phagocytic cells, granulocytes, and solublemacromolecules produced by the above cells or the liver (includingantibodies, cytokines, and complement) that results in selective damageto, destruction of, or elimination from the human body of cancerouscells, metastatic tumor cells, invading pathogens, cells or tissuesinfected with pathogens, or, in cases of autoimmunity or pathologicalinflammation, normal human cells or tissues. Components of an immuneresponse can be detected in vitro by various methods that are well knownto those of ordinary skill in the art.

Generation of an immune response by the Tymovirus virus or TymovirusVLPs is typically undesirable. Accordingly, in some embodiments it maybe preferable to modify the Tymovirus virus or Tymovirus VLPs or takeother steps to decrease the immune response. For example, animmunosuppressant compound can be administered to decrease the immuneresponse. More preferably, the Tymovirus virus or Tymovirus VLPs can bemodified to decrease its immunogenicity, such as through the use ofshielding molecules that prevent immune clearance. Examples of methodssuitable for decreasing immunity include attachment of anti-fouling(e.g., zwitterionic) polymers, glycosylation of the virus carrier, andPEGylation.

In some embodiments, the immunogenicity of the Tymovirus virus orTymovirus VLPs are decreased by PEGylation. PEGylation is the process ofcovalent attachment of polyethylene glycol (PEG) polymer chains to amolecule, such as a Tymovirus VLP. PEGylation can be achieved byincubation of a reactive derivative of PEG with the Tymovirus virus orTymovirus VLP. The covalent attachment of PEG to the Tymovirus virus orTymovirus VLP can “mask” the agent from the host's immune system, andreduce production of antibodies against the carrier. PEGylation also mayprovide other benefits. PEGylation can be used to vary the circulationtime of the Tymovirus virus or Tymovirus VLPs. For example, use of PEG5,000 can provide a virus-like particle with a circulation half-lifemuch lower than the use of PEG 20,000.

The first step of PEGylation is providing suitable functionalization ofthe PEG polymer at one or both terminal positions of the polymer. Thechemically active or activated derivatives of the PEG polymer areprepared to attach the PEG to the Tymovirus virus or Tymovirus VLPs.There are generally two methods that can be used to carry outPEGylation; a solution phase batch process and an on-column fed-batchprocess. The simple and commonly adopted batch process involves themixing of reagents together in a suitable buffer solution, preferably ata temperature between 4° and 6° C., followed by the separation andpurification of the desired product using a chromatographic technique.

Administration and Formulation of Tymovirus Virus or Tymovirus VLPs

In some embodiments, a plurality of Tymovirus virus or Tymovirus VLPsare administered together with a pharmaceutically acceptable carrier toprovide a pharmaceutical formulation. Pharmaceutically acceptablecarriers enable the Tymovirus virus or Tymovirus VLPs to be delivered tothe subject in an effective manner while minimizing side effects, andcan include a variety of diluents or excipients known to those ofordinary skill in the art. Formulations include, but are not limited to,those suitable for oral, rectal, vaginal, topical, nasal, ophthalmic, orparental (including subcutaneous, intramuscular, intraperitoneal,intratumoral, and intravenous) administration. For example, forparenteral administration, isotonic saline is preferred. For topicaladministration, a cream, including a carrier such as dimethylsulfoxide(DMSO), or other agents typically found in topical creams that do notblock or inhibit activity of the compound, can be used. Other suitablecarriers include, but are not limited to, alcohol, phosphate bufferedsaline, and other balanced salt solutions.

The formulations may be conveniently presented in unit dosage form andmay be prepared by any of the methods well known in the art of pharmacy.Preferably, such methods include the step of bringing the Tymovirusvirus or Tymovirus VLPs into association with a pharmaceuticallyacceptable carrier that constitutes one or more accessory ingredients.In general, the formulations are prepared by uniformly and intimatelybringing the active agent into association with a liquid carrier, afinely divided solid carrier, or both, and then, if necessary, shapingthe product into the desired formulations. The methods of the inventioninclude administering to a subject, preferably a mammal, and morepreferably a human, the composition of the invention in an amounteffective to produce the desired effect. The formulated functionalizedTymovirus virus or Tymovirus VLPs can be administered as a single doseor in multiple doses.

Useful dosages of the active agents can be determined by comparing theirin vitro activity and the in vivo activity in animal models. Methods forextrapolation of effective dosages in mice, and other animals, to humansare known in the art; for example, see U.S. Pat. No. 4,938,949. Anamount adequate to accomplish therapeutic or prophylactic treatment isdefined as a therapeutically- or prophylactically-effective dose. Inboth prophylactic and therapeutic regimes, agents are usuallyadministered in several dosages until an effect has been achieved.Effective doses of the Tymovirus virus or Tymovirus VLPs vary dependingupon many different factors, including means of administration, targetsite, physiological state of the patient, whether the patient is humanor an animal, other medications administered, and whether treatment isprophylactic or therapeutic.

The dosage of an imaging agent, therapeutic agent, and/or targetingagent including in a plurality of Tymovirus virus or Tymovirus VLPs foradministration to a mammalian subject or an avian subject in accordancewith a method described herein ranges from about 0.0001 to 100 mg/kg,and more usually 0.01 to 5 mg/kg, of the host body weight. For exampledosages can be 1 mg/kg body weight or 10 mg/kg body weight or within therange of 1-10 mg/kg. A suitable amount of Tymovirus virus or TymovirusVLPs are used to provide the desired dosage of agent(s). An exemplarytreatment regime entails administration once per every two weeks or oncea month or once every 3 to 6 months. The plurality of Tymovirus virus orTymovirus VLPs are usually administered on multiple occasions.Alternatively, the Tymovirus virus or Tymovirus VLPs can be administeredas a sustained release formulation, in which case less frequentadministration is required. In therapeutic applications, a relativelyhigh dosage at relatively short intervals is sometimes required untilprogression of the disease is reduced or terminated, and preferablyuntil the patient shows partial or complete amelioration of symptoms ofdisease. Thereafter, the patient subject can be administered aprophylactic regime.

Compositions including Tymovirus virus or Tymovirus VLPs describedherein can also include, depending on the formulation desired,pharmaceutically-acceptable, nontoxic carriers or diluents, which aredefined as vehicles commonly used to formulate pharmaceuticalcompositions for animal or human administration. The diluent is selectedso as not to affect the biological activity of the combination. Examplesof such diluents are distilled water, physiological phosphate-bufferedsaline, Ringer's solutions, dextrose solution, and Hank's solution. Inaddition, the pharmaceutical composition or formulation may also includeother carriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenicstabilizers and the like.

Pharmaceutical compositions can also include large, slowly metabolizedmacromolecules such as proteins, polysaccharides such as chitosan,polylactic acids, polyglycolic acids and copolymers (such as latexfunctionalized SEPHAROSE, agarose, cellulose, and the like), polymericamino acids, amino acid copolymers, and lipid aggregates (such as oildroplets or liposomes).

For parenteral administration, compositions of the invention can beadministered as injectable dosages of a solution or suspension of thesubstance in a physiologically acceptable diluent with a pharmaceuticalcarrier that can be a sterile liquid such as water oils, saline,glycerol, or ethanol. Additionally, auxiliary substances, such aswetting or emulsifying agents, surfactants, pH buffering substances andthe like can be present in compositions. Other components ofpharmaceutical compositions are those of petroleum, animal, vegetable,or synthetic origin, for example, peanut oil, soybean oil, and mineraloil. In general, glycols such as propylene glycol or polyethylene glycolare preferred liquid carriers, particularly for injectable solutions.

In other embodiments, Tymovirus virus or Tymovirus VLPs can beadministered to a subject in a method of detecting cancer. The methodincludes administering a plurality of functionalized Tymovirus virus orTymovirus VLPs that have been loaded with or conjugated to an imagingagent. The method also includes detecting the imaging agent in thesubject using an imaging device subsequent to administering theTymovirus virus or Tymovirus VLPs to determine the location and/ordistribution of the cancer in the subject. The cancer can be detected ina particular area or portion of the subject and, in some instances, twoor more areas or portions throughout the entire subject. For example,the cancer can be detected in diseased tissue, including neoplastic orcancerous tissue (e.g., tumor tissue). The cancer can include a solidtumor, such as a solid carcinoma, sarcoma or lymphoma. The tumor caninclude both cancerous and pre-cancerous cells. The detected cancer caninclude malignant cancer metastases.

The particular area or portion of the subject can include regions to beimaged for both diagnostic and therapeutic purposes. The particularcancerous area or portion of the subject where cancer is detected istypically internal; however, it will be appreciated that the cancer mayadditionally or alternatively be external.

At least one image of the particular cancerous area or portion of thesubject can be generated using an imaging agent once the Tymovirus virusor Tymovirus VLPs localize to the cancer. The imaging agent can includeone or combination of known imaging techniques capable of visualizingthe virus or VLPs. Examples of imaging modalities can include ultrasound(US), magnetic resonance imaging (MRI), nuclear magnetic resonance(NMR), computed topography (CT), electron spin resonance (ESR), nuclearmedical imaging, optical imaging, and positron emission topography(PET).

In one example, the imaging agents loaded with or conjugated to thevirus or VLPs can be detected with MRI and/or x-ray. MRI relies uponchanges in magnetic dipoles to perform detailed anatomic imaging andfunctional studies.

Optionally, the Tymovirus virus or Tymovirus VLPs can be modified tofacilitate detection and imaging with MRI and CT as well as positronemission tomography (PET). For MRI applications, gadolinium tags can beattached to the conjugated to or loaded in the VLPs. For PETapplications, radioactive tags can be attached to virus or VLPs. For CTapplications, iodide or other heavy metals can be attached to theTymovirus virus or Tymovirus VLPs to facilitate CT contrast.

It will be appreciated that the Tymovirus virus or Tymovirus VLPs willlikely be most useful clinically when several imaging techniques orimaging followed by a medical or surgical procedure is used. In thisway, the ability to use one agent for multiple imaging modalities isoptimized making the Tymovirus virus or Tymovirus VLPs cost-competitivewith existing contrast agents.

For multimodal imaging applications, the Tymovirus virus or TymovirusVLPs can be administered to a subject and then preoperatively imagedusing, for example, CT or MRI. After preoperative imaging, the Tymovirusvirus or Tymovirus VLPs can serve as optical beacons for use duringsurgery leading to more complete resections or more accurate biopsies.In surgical resection of lesions, the completeness of resection can beassessed with intra-operative ultrasound, CT, or MRI. For example, inprostate cancer or breast cancer surgery, the Tymovirus virus orTymovirus VLPs can be given intravenously about 24 hours prior topre-surgical stereotactic localization MRI. The viral nanoparticles canbe imaged on gradient echo MRI sequences as a contrast agent thatlocalizes with a prostate or breast cancer tumor.

The present invention is illustrated by the following examples. It is tobe understood that the particular examples, materials, amounts, andprocedures are to be interpreted broadly in accordance with the scopeand spirit of the invention as set forth herein.

EXAMPLE Physalis Mottle Virus-Like Particles as Nanocarriers for ImagingReagents and Drugs

In this example, we describe VLPs based on Physalis mottle virus (PhMV)and their use for imaging and drug delivery. PhMV (Tymovirus,Tymoviridae) has an about 30-nm icosahedral capsid with T=3 symmetry,containing a single-stranded, plus-sense RNA genome of 6.67 kb. Thegenome is encapsidated in a protein shell comprising 180 chemicallyidentical 21 kDa coat protein subunits, with three distinct bondingpatterns (A, B and C). The A type subunits form pentamers at theicosahedral five-fold axes (60 subunits), whereas the B and C typesubunits form 20 hexamers at the icosahedral three-fold axes (120subunits). The protein subunits are held in place by strongprotein-protein interactions. The multiple copies of the asymmetric unitprovide regularly spaced attachment sites on both the internal andexternal surfaces of the PhMV capsid. The PhMV coat protein expressed inE. coli was shown to self-assemble into stable VLPs that were nearlyidentical to the viruses formed in vivo. These VLPs can be purified inlarge quantities (50-100 mg/L) and are exceptionally robust, maintainingtheir integrity within the pH range 4.2-9.0 and in the presence of up to5 M urea. They are monodisperse, symmetrical and polyvalent. Neither thedeletion nor the addition of amino acids at the N-terminus of the PhMVcoat protein hinders capsid assembly, making this an ideal site formodifications. The three-dimensional crystal structures of PhMV and itsempty capsid have been determined to 3.8 and 3.2 Å resolution,respectively. The structures indicate that the empty shells correspondto a “swollen state” of the virus, with increased disorder in theN-terminal segments as well as some positively charged side chainslining the internal cavity.

PhMV-derived VLPs have been genetically engineered to display diagnosticand immunogenic epitopes. PhMV offers the following advantages:

(1) The genome is small (2) It is easy to manipulate (3) Purification issimple and quicker than the regeneration of stably transformed plants.Coat protein of PhMV expresses extremely well as empty capsids in E.coli resulting in (1) Yields as high as 100-150 irmg per liter ofculture, (2) Batch to batch variations are nil as each and every timeconfirmation of the assembled capsids will be maintained in a similarway for the integrity of the capsids, (3) Recombinant Ph-CP is stableover a wide range of pH from 4.2 to 9.0 and stable upto 4 M urea, (4)Purification of empty capsids is easy (5) Mechanism of assembly of emptycapsids is well studied. (6) Host range is very narrow.

Here, we show the internal and external surface chemistries of PhMV anddeveloped protocols to achieve the specific functionalization of thesesurfaces with different reagents. A library of functionalizationprotocols, including bioconjugation and non-covalent infusion, was usedto modify PhMV with dyes, drugs and photosensitizers. Thesefunctionalized PhMV nanoparticles were then characterized by flowcytometry and confocal microscopy, and their cytotoxic efficacy wastested in a range of normal and cancer-derived cell lines.

Methods Expression and Purification of PhMV VLPs

The 564-bp PhMV coat protein gene (GenBank S97776) was prepared as asynthetic construct by Invitrogen GeneArt, with XhoI and HindIII sitesat the 5′ and 3′ ends, respectively, for insertion into pRSET-A(Invitrogen) at the same sites. The integrity of the recombinant vector(pR-PhMV-CP) was confirmed by automated DNA sequencing before thetransformation of ClearColi BL21(DE3) cells. The expression andpurification of PhMV VLPs is described. Briefly, a single colonycarrying pR-PhMV-CP was inoculated into 50 mL of lysogeny broth (LB)containing 100 mg/mL ampicillin and was incubated for 20 h at 37° C. Wethen used 5 mL of the pre-culture to inoculate 500 mL LB with ampicillinas above. After 4-5 h of growth (OD_(600 nm)=0.6), expression wasinduced with 0.5 mM IPTG and the cells were incubated at 30° C.overnight. The culture was centrifuged (6000 rpm, 10 min, 4° C.) and thepellet was suspended in 50 mM sodium citrate buffer, pH 5.5 (SCB). Thesuspension was then sonicated and centrifuged at 35,000 rpm using a 50.2Ti rotor (Beckman Coulter Inc.) at 4° C. for 3 h. The pellet wasresuspended in SCB and layered onto a 10-40% linear sucrose gradient andcentrifuged at 28,000 rpm in an SW 32 Ti rotor (Beckman Coulter Inc.) at4° C. for 3 h. The light scattering zone was collected with a Pasteurpipette, diluted with SCB and centrifuged at 42,000 rpm using 50.2 Tirotor at 4° C. for 3 h. The final pellet was resuspended in SCB andstored at 4° C. The protein concentration was measured using Bradfordreagent (BioRad).

Bioconjugation Reactions

External lysine residues were conjugated to sulfo-Cy5 NHS ester(Lumiprobe), whereas internal cysteine residues were conjugated toCy5.5-maleimide (Lumiprobe). The dyes were added to PhMV at aconcentration of 1 mg/mL in KP buffer (0.01 M potassium phosphate bufferpH 7.0) at a molar excess of 900 Cy5 molecules per particle (fivemolecules per coat protein) and 360 Cy5.5 per particle (two moleculesper coat protein). The final DMSO concentration was adjusted to 10%(v/v). The reaction was left for 2 h (Cy5) or overnight (Cy5.5) at roomtemperature with agitation in the dark. Both reaction mixtures werepurified over a 30% (w/v) sucrose cushion by ultracentrifugation at52,000 rpm using a TLA55 rotor (Beckman Coulter Inc.) for 1 h. Pelletscontaining dye-labeled particles were resuspended in KP buffer overnightat 4° C. For PhMV-K_(E)-Cy5 particles, 10 kDa molecular weight cutoff(MWCO) centrifugal filters (Amicon) were also used to remove excess dyemolecules. Any aggregates were removed by a clearing spin at 12000 rpmfor 10 min using a table-top centrifuge. For the biotin conjugationreactions, the VLPs were used at a final concentration of 1 mg/mL in KPbuffer and were incubated with a 360-fold molar excess of biotin(biotin-NHS ester or biotin-maleimide) at room temperature overnight,with agitation. The final DMSO concentration was adjusted to 10% of thereaction volume. Particles were purified using 10 kDa MWCO centrifugalfilter units (Millipore).

Infusion Protocol

The VLPs were loaded with rhodamine B, fluorescein, crystal violet, MTXdihydrochloride (all Sigma-Aldrich), PS (cationic zinc ethynylphenylporphyrin, full name5-(4-ethynylphenyl)-10,15,20-tris)4-methylpyridin-4-ium-1-yl)porphyrinzinc(II) triiodide), or DOX hydrochloride (Indofine Chemical Company).The VLPs (1 mg/mL in KP buffer) were incubated with a molar excess of500, 2500, 5000, or 10000 cargo molecules per particle overnight at roomtemperature in the dark, before purification over a 30% (w/v) sucrosecushion to remove excess reagents by ultracentrifugation at 52,000 rpmusing a TLA 55 rotor (Beckman Coulter Inc.) at 4° C. for 1 h. PS-PhMVand DOX-PhMV were synthesized for further characterization using a5000-fold molar excess.

UV/Visible Spectroscopy

A NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific) was used tocharacterize the UV/visible spectra of native and modified VLPs. The dyeload was determined using the protein concentration (measured using theBradford assay), the Beer-Lambert law and the following dye-specificextinction coefficients: rhodamine B, ε(553 nm)=116,000 M⁻¹ cm⁻¹; DOX,ε(496 nm)=11,500 M⁻¹ cm⁻¹; crystal violet, ε(590 nm)=87,000 M⁻¹ cm⁻¹;PS, ε(450 nm)=195,000 M⁻¹ cm¹; MTX, ε(622 nm)=25,000 M⁻¹ cm¹; sulfo-Cy5NHS ester, ε(646 nm)=271,000 M⁻¹ cm¹; Cy5.5-maleimide, 8(673 nm)=209,000M⁻¹ cm⁻¹. The following molecular weights were used: PhMV=4.7×10⁶ gmol⁻¹; rhodamine B=479.02 g mol⁻¹; DOX=579.98 g mol⁻¹; crystalviolet=407.98 g mol¹; PS=1130 g mol⁻¹; mitoxantrone=517.4 g mol⁻¹;Cy5=777.95 g mol⁻¹; Cy5.5=741.36 g mol⁻¹; Biotin NHS ester=341.38 gmol⁻¹; Biotin maleimide=451.54 g mol⁻¹.

Native and Denaturing Gel Electrophoresis

Intact VLPs (10-20 μg per lane) were analyzed by 1% (w/v) agarose nativegel electrophoresis in 0.1 M Tris-maleate running buffer (pH 6.5) aspreviously described. Denatured protein subunits (10 μg per lane) wereanalyzed by polyacrylamide gel electrophoresis using 4-12% NuPAGE gelsand 1×MOPS buffer (Invitrogen). Samples were denatured by boiling in SDSloading dye for 10 min. Gels were photographed under UV or white lightbefore staining with Coomassie Blue, and under white light afterstaining, using an AlphaImager system (Biosciences).

Size Exclusion Chromatography

VLPs were analyzed by SEC using a Superose-6 column on the AkTA Explorersystem (GE Healthcare). The column was loaded with 100 μL samples (1mg/mL) at a flow rate of 0.5 mL min⁻¹ in KP buffer.

Transmission Electron Microscopy

VLPs suspended at 1 mg/mL in 20 μL KP buffer were deposited onto Formvarcarbon film coated copper TEM grids (Electron Microscopy Sciences) for 2min at room temperature. The grids were then washed twice with deionizedwater for 45 s and stained twice with 2% (w/v) uranyl acetate indeionized water for another 30 s. A Tecnai F30 transmission electronmicroscope was used to analyze the samples at 300 kV.

Zeta Potential Analysis

The zeta potential (ζ) of the VLPs was determined by placing 0.25 mg/mLsolutions of each VLP in a 90Plus Zeta potential analyzer (BrookhavenInstruments) and conducting five measurements, each comprising six runs.

Avidin Agarose Affinity Binding Assay

Biotinylated VLPs and controls were tested for their ability to bindavidin agarose resin (Pierce). The batch method provided by the supplierwas used, with some modifications: 100 μg samples in 100 μL bindingbuffer (PBS with 0.1% SDS and 1% NP-40) were added to 100 μL of theresin, and the resulting 200 μL of slurry was mixed for 1 h at roomtemperature. The supernatant was then recovered and the resin washed sixtimes in 100 μL binding buffer. Bound VLPs were eluted in 100 μL 0.1 Mglycine-HCl buffer (pH 2.8) and the pH was immediately adjusted with 10μL 1 M Tris buffer (pH 7.5). Samples of the wash fractions and theeluate were analyzed by denaturing gel electrophoresis, 30 μL per lane.

Tissue Culture

All cell lines were obtained from the American Type Culture Collection(ATCC). HeLa (cervical cancer), RAW264.7 (leukemic macrophages), A2780(ovarian cancer), MDA-MB-231 (breast cancer) and U87 (brain cancer) celllines were maintained in Dulbecco's modified Eagle's medium (DMEM)supplemented with 10% (v/v) fetal bovine serum (FBS, AtlantaBiologicals), 1% (w/v) penicillin/streptomycin (pen/strep, Thermo FisherScientific) and 1% (w/v) glutamine, at 37° C. and 5% CO₂. HT1080(fibrosarcoma) cells were maintained in Minimum Essential Medium (MEM)supplemented with 10% (v/v) FBS, 1% (w/v) pen/strep and 1% (w/v)glutamine as above. PC-3 cells (prostate cancer) were maintained inRoswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10%(v/v) FBS, 1% (w/v) pen/strep and 1% (w/v) glutamine as above. NIH3T3murine fibroblasts were maintained in DMEM/F12 medium containing 10%(v/v) newborn calf serum, 1% (w/v) pen/strep and 1% (w/v) GlutaMax asabove.

Confocal Microscopy

Cell lines were grown for 24 h on glass coverslips (25,000 cells perwell) placed in an untreated 24-well plate in 200 μL of the appropriatemedium. The cells were washed twice with Dulbecco's PBS (DPBS) beforeadding the PhMV-K_(E)-Cy5 or PhMV-C_(I)-Cy5.5 particles (2.5×10⁶particles per cell, corresponding to ˜0.5 μg/well) and incubating for 6h. The cells were washed twice in DPBS to remove unbound particles andfixed for 5 min at room temperature in DPBS containing 4% (v/v)paraformaldehyde and 0.3% (v/v) glutaraldehyde. Cell membranes werestained with 1 μg/mL wheat germ agglutinin conjugated to AlexaFluor-555(Invitrogen) diluted 1:1000 in DPBS containing 5% (v/v) goat serum, andthe cells were then incubated for 45 min at room temperature in thedark. Finally, the cells were washed thrice with DPBS, and thecoverslips were mounted on glass slides using Fluroshield with 4′,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich) and sealed using nailpolish. Confocal images were captured on a Leica TCS SPE confocalmicroscope and the images were processed using Image J v1.44o.

For co-localization studies, A2780 and MDA-MB-231 cells were incubatedfor 6 h with PhMV-C_(I)-Cy5.5 particles, then blocked in 10% (v/v) goatserum for 45 min to reduce non-specific antibody binding. Endolysosomeswere stained using a mouse anti-human LAMP-1 antibody (Biolegend)diluted 1:250 in 5% goat serum for 60 min, with DAPI staining andimaging as described above. VLPs were visualized by detecting thecovalently-attached Cy5.5 maleimide dye as described above.

Fluorescence-Activated Cell Sorting

Cells were grown to confluency, collected in enzyme-free Hank's-basedcell dissociation buffer, and distributed in 200 μL aliquots at aconcentration of 2×10⁵ cell/mL in V-bottom 96-well plates. Dye-labeledVLPs (100,000 particles per cell, corresponding to ˜1.6 μg/well) wereadded to the cells and incubated for 6 h. The cells were washed twice inFACS buffer (0.1 mL 0.5 M EDTA, 0.5 mL FBS, and 1.25 mL 1 M HEPES, pH7.0 in 50 mL Ca²⁺ and Mg²⁺ free PBS) and fixed in 2% (v/v) formaldehydein FACS buffer for 10 min at room temperature. Cells were washed andresuspended in FACS buffer and analyzed using a BD LSR II flowcytometer. Triplicates of each sample were maintained and at least10,000 events (gated for live cells) were recorded. Data were analyzedusing FlowJo v8.6.3.

LIVE/DEAD Assay

PC-3 cells were seeded (20,000 cells/500 L RPMI/well) in a 24-well plateovernight. The cells were washed twice in PBS and incubated for 8 h intriplicates with 5.0 μM PS, 5.0 μM PS-PhMV, or the correspondingconcentration of unloaded VLPs (236.70 M or ˜0.19 mg/mL). After washingtwice in PBS, 500 μL RPMI medium was added. Photodynamic therapy wasthen applied using a white light source (Phillips Silhouette High OutputF39T5/841 HO, Alto collection, ˜10 mW cm⁻²) for 30 min (18.1 J cm⁻² at430 nm) and cells were incubated for a further 48 h in the dark. Cellviability was determined using a LIVE/DEAD assay for mammalian cells(Thermo Fisher Scientific) following the manufacturer's procedures forcell staining, and cells were observed under a Zeiss Axio Observer Z1motorized FL inverted microscope.

MTT Cell Viability Assay

PC-3 cells were seeded (5000 cells/100 μL RPMI 1640/well) in a 96-wellplate overnight. After two PBS washes to remove unbound and dead cells,free PS, or PS-PhMV was added to the cells in triplicate atconcentrations of 0.01, 0.025, 0.05, 0.1, 0.25, 0.5, 1, 2.5 and 5.0 μMPS, and incubated for 8 h. Untreated cells and cells treated withunloaded VLPs at the equivalent protein concentration to PS-PhMVparticles at the highest dose of PS were also used as controls. Freeparticles were washed with PBS and 100 μL of fresh medium was added. Thecells were then illuminated with white light for 30 min as above. Inparallel, a duplicate plate was prepared and kept in the dark as anegative control. Following phototherapy, cells were incubated foranother 48 h in the dark, and their viability was subsequently measuredusing an MTT cell proliferation assay kit (ATCC) based on themanufacturer's instructions. A Tecan Infinite 200 PRO multimode platereader was used to measure absorbance at 570 nm, and the percent cellviability was normalized to the untreated control. All assays werecarried out at least three times. The efficacy of DOX-PhMV was tested byseeding A2780 and MDA-MB-231 cells as above (5000 cells/100 μLDMEM/well) and treating triplicate wells with free DOX or DOX-PhMV atconcentrations of 0.01, 0.05, 0.1, 0.5, 1, 5.0 and 10.0 μM DOX for 24 h.Untreated cells and cells treated with unloaded VLPs at a concentrationequivalent to the highest dose of DOX-PhMV were used as controls.Washing steps and the MTT assay were then carried out as above.

Results Purification and Characterization of PhMV-Derived VLPs Producedin ClearColi Cells

PhMV-derived VLPs were purified from ClearColi cells to avoid endotoxincontamination. The coat protein gene (564 bp) was inserted into thevector pRSET-A and expressed in ClearColi BL21(DE3). Optimum expressionwas achieved by inducing the culture with 0.5 mM isopropylβ-D-1-thiogalactopyranoside (IPTG) and cultivating at 30° C. overnight.SDS-PAGE analysis confirmed the presence of the coat protein in totaland soluble cell protein extracts and VLPs were purified from thesoluble fraction. A single light-scattering zone was observed in a10-40% (w/v) linear sucrose density gradient and the VLP yield was 40-50mg per liter of culture medium, as determined using the Bradford assay.

The denatured VLP preparation revealed a single 26 kDa bandcorresponding to the coat protein. Fast protein liquid chromatography(FPLC) analysis confirmed that the particles eluted as a single peak at7.5 mL, indicating they were intact and stable (Figure S1D).Transmission electron microscopy (TEM) revealed that the VLPs wereapproximately spherical and 29±2 nm in diameter, indicating thatrecombinant PhMV coat proteins were indeed capable of self-assembly. Thezeta potential of the VLPs was +4.20±0.46 (Table 1).

TABLE 1 Zeta potential measurement of functionalized PhMV Zeta potentialSample (Standard error in parenthesis) Native PhMV +4.20 (0.46)PhMV-K_(E) Cy5 −7.92 (2.49) PhMV-C_(I) Cy5.5 +0.38 (3.32) DOX-PhMV +9.38(1.93) PS-PhMV −0.81 (3.08)

Structure-Based Design of PhMV-Derived VLPs to Carry Dyes and Drugs

A reliable PhMV-based platform for chemical modification requires theidentification of attachment sites on the capsid that do not compromisethe structure of the asymmetric unit and its native biologicalfunctions, but nevertheless allow for efficient bioconjugationreactions. The bioconjugation sites on the internal and externalsurfaces of the VLP were chosen by studying the structural contributionof each residue. Nine lysine residues are present on each PhMV coatprotein subunit, four of which (K62, K143, K153, and K166) are exposedon the exterior, resulting in 720 addressable lysine residues per VLPthat can be used for bioconjugation based on lysine/N-hydroxysuccinimide(NHS) ester chemistry (FIG. 1A). The coat protein contains only a singlecysteine residue (C75) and this is presented on the internal surface,resulting in 180 addressable cysteine residues per VLP potentiallysuitable for bioconjugation using thiol-maleimide chemistry (FIG. 1A).As well as the development of bioconjugation protocols (FIGS. 1B, C), wealso considered the encapsulation of cargo molecules in the cavity. Theencapsulation of dyes and drugs via particle disassembly and assembly isnot possible in the case of PhMV because the virus is stabilizedpredominantly by protein-protein interactions. We therefore developed aninfusion protocol to load the VLPs with cargo (FIGS. 1D-E).

Bioconjugation of PhMV-Derived VLPs with Dyes

Surface-exposed lysine residues were conjugated to NHS-activated estersof sulfo-cyanine 5 succinimidyl ester (sulfo-Cy5) by incubating for 2 hwith a 900-fold molar excess of the dye, equivalent to five dyemolecules per coat protein (FIG. 1B). Similarly, the thiol groups on theinternal cysteine residues were conjugated overnight usingCy5.5-maleimide at a 360-fold molar excess, equivalent to two dyemolecules per coat protein (FIG. 1C). The resulting VLP-dye conjugates(PhMV-K_(E)-Cy5 and PhMV-C_(I)-Cy5.5, where K_(E)=external lysine andC_(I)=internal cysteine) were purified by ultracentrifugation, andPhMV-K_(E)-Cy5 was purified further by ultrafiltration to remove freedye molecules. The Bradford protein assay was used to estimate theconcentration of PhMV-K_(E)-Cy5 and PhMV-C_(I)-Cy5.5 particles.UV/visible spectroscopy was used to determine the number of dyemolecules per particle based on the Beer-Lambert law and dye-specificextinction coefficients, revealing that the PhMV-K_(E)-Cy5 particlescontained 160-180 Cy5 molecules and the PhMV-C_(I)-Cy5.5 particlescontained 40-60 Cy5.5 molecules. The higher labeling efficiency achievedusing lysine-NHS chemistry was expected due to the presence of 720surface-exposed lysine residues compared to 180 internal cysteineresidues.

Increasing the molar excess of Cy5.5-maleimide did not increase theinternal labeling density, and maximum labeling efficiency was achievedwith two dye molecules per coat protein. In contrast, we achieved agreater density of external labeling when the molar excess of sulfo-Cy5was increased to 20 dye molecules per coat protein but further increaseswere not tested because the dye aggregated at higher concentrations. Forimaging applications, the spatial distribution of dye molecules isimportant, so we measured the distance between the surface-exposedlysine side chains using Chimera software and found the spacing liesbetween 1-5 nm. However, if we assume random distribution of the Cy5molecules conjugated to the external lysine side chains, the averagedistance between two fluorophores would be ˜2-4 nm assuming 1-2 dyes percoat protein. Based on the Foerster radius, which suggests quenchinggenerally occurs when dyes are <10 nm apart, we would expect quenchingto occur for the labeled VLPs due to cross-talk between the fluorophorecenters. Nevertheless, detection was not an issue for these particles.Furthermore, when taken up by cells, the dyes are on these particles arelikely to be cleaved from the VLP as has been previously shown. Furtherstructure-function studies would be required for the development offluorophore-labeled PhMV VLPs as optical probes.

We characterized the PhMV-K_(E)-Cy5 and PhMV-C_(I)-Cy5.5 particles usinga combination of native and denaturing gel electrophoresis, zetapotential analysis, FPLC, and TEM. Native and denaturing gelelectrophoresis followed by visualization under white light beforeCoomassie staining confirmed the covalent attachment of the dyes (FIGS.2A, B; lanes 2, 3). The charge of the PhMV-K_(E)-Cy5 particles wasaltered following bioconjugation, as evident from the mobility shifttowards the anode during native electrophoresis (FIG. 2B, lane 2). Thiswas anticipated because the addition of the non-charged dye removespositive amines from the particle surface and thus reduces the overallpositive surface charge. Accordingly, zeta potential measurementsrevealed that the PhMV-K_(E)-Cy5 particles were negatively charged(−7.92) whereas native PhMV particles are positively charged (+4.20)(Table 1). In contrast, the mobility of the PhMV-C_(I)-Cy5.5 particleswas the same as wild-type PhMV because cysteine residues are uncharged(FIG. 2B, lane 3). Zeta potential measurements indicated a reduction inthe net positive charge of the PhMV-C_(I)-Cy5.5 particles (+0.38),probably reflecting changes in surface charge distribution.

FPLC and TEM analysis indicated that the dye-labeled VLPs were intact.Dye-labeled particles eluted as a single peak from the Superose-6 columnat an elution volume of 7.5 mL, the same as wild-type particles, and thefluorophore co-eluted with the PhMV-K_(E)-Cy5 and PhMV-C_(I)-Cy5.5particles at 646 nm and 673 nm, respectively (FIG. 2C; top panel). TEManalysis revealed that the labeled particles remained monodisperse, witha diameter of 28-29 nm based on ImageJ analysis (FIG. 2D; top panel).Finally, the labeled particles remained stable when stored at 4° C. inKP buffer (0.01 M potassium phosphate, pH 7.0) for several months.

Spatial Distribution of Biotin in VLP-Biotin Conjugates

To confirm the position of the bioconjugation sites, we modified eachsite with biotin labels and mapped them using an avidin bead assay. Thiswas necessary because although maleimide chemistry is thiol selective,some reports indicate cross-reactivity with lysine residues. Externallysine residues were therefore labeled using a 360-fold molar excess ofa NHS-reactive biotin probe to generate PhMV-K_(E)-bio particles, andinternal cysteine residues were labeled using a 360-fold molar excess ofa maleimide-reactive biotin probe to generate PhMV-C_(I)-bio particles.The resulting particles were analyzed by native and denaturing gelelectrophoresis. As expected, we observed no difference in mobilitybetween labeled and unlabeled particles in the denaturing gels (FIG.3A), whereas the mobility of PhMV-K_(E)-bio but not PhMV-C_(I)-biodiffered from the unlabeled particles in the native gels due to theelimination of positive surface charges, consistent with the behavior ofthe dye-labeled particles above (FIG. 3B).

The avidin bead assay was used to selectively capture VLPs displayingbiotin on the external surface. The avidin agarose beads were mixed withunlabeled VLPs, PhMV-K_(E)-bio particles, or PhMV-C_(I)-bio particles;the beads were washed, and the particles were eluted in agglutinin,which stains the cell membrane. The analysis of confocal images usingImageJ software confirmed the internalization of PhMV-C_(I)-Cy5.5particles. In A2780 and MDA-MB-231 cells, we confirmed that theinternalization of PhMV-C_(I)-Cy5.5 particles involved endocytosis, andthe particles colocalized with the endolysosomal marker LAMP-1 (FIG.4C).

These results indicate that PhMV-C_(I)-Cy5.5 particles are the mostsuitable candidates for future in vivo imaging and tumor homing studies.The efficient internalization of these particles by cancer cells (up to100% uptake) probably reflects the retained positive surface charge(+0.38) as revealed by native gel electrophoresis (FIG. 2B, lane 3). Incontrast, PhMV-K_(E)-Cy5 particles are taken up less efficiently due tothe neutralization of positively charged lysine residues by NHSesterification (FIG. 2B, lower panel, lane 2), resulting in a netnegative charge of −7.92 that would repel like charges on the cellmembrane.

VLP Infusion and Characterization of the Loaded Particles

The potential of the VLPs to encapsulate cargo molecules was testedusing the tracer dye rhodamine B, fluorescein, the cancer drugdoxorubicin (DOX), the anthelmintic drug crystal violet, thephotodynamic therapeutic photosensitizer (PS) cationic zincethynylphenyl porphyrin, and the cancer drug mitoxantrone (MTX), all ofwhich are positively charged and fluorescent—apart from fluoresceinwhich is neutral, allowing quantification by UV/visible spectroscopybased on absorbance.

Intact VLPs were incubated in a bathing solution containing the guestmolecule at various molar excesses (500, 2500, 5000 and 10,000 moleculesper VLP) in KP buffer with 10% (v/v) DMSO overnight at room temperature.After the reaction, excess guest molecules were removed byultracentrifugation and the amount of protein and cargo was quantifiedby the Bradford assay and UV/visible spectroscopy, respectively. Weapplied the Beer-Lambert law and the specific extinction coefficient ofeach guest molecule to determine the number of cargo molecules per VLP(FIG. 5). The VLPs showed the greatest affinity for crystal violet (2000molecules per particle) and the lowest affinity for rhodamine B,fluorescein, and PS (˜200 molecules per particle). The VLPs showedintermediate affinities for DOX and MTX, with 750 DOX and 450 MTXmolecules per particle, respectively (FIG. 5). The differences inloading probably reflect the density and distribution of charged andhydrophobic groups on the guest molecules.

The purification of the drug-loaded VLPs resulted in 50-60% recoverycompared to the amount of starting material. Two formulations (PS-PhMVand DOX-PhMV) were taken forward for complete characterization andtesting for in vitro cargo delivery and cell killing efficacy. We used a5000-fold molar excess of PS or DOX per particle for further experimentsbecause this facilitated efficient loading without significantaggregation. Extending the incubation time did not increase the loadingefficiency at this molar ratio (data not shown). We consistentlyobserved the loading of 160-180 PS molecules and 600-800 DOX moleculesper VLP (FIG. 5).

Denaturing gel electrophoresis was used to confirm that PS and DOX wereloaded into the cavity of the VLPs non-covalently (FIGS. 2A, B). Gelswere visualized under UV light before Coomassie staining, and underwhite light afterwards. In denaturing gels, the PS-PhMV and DOX-PhMVcoat proteins co-migrated with the native PhMV coat protein. Denaturingreleases the encapsulated cargo (PS or DOX), which has a high mobilitydue to the low molecular weight of each molecule (PS=1130 g mol⁻¹,DOX=579.98 g mol⁻¹) resulting in a fluorescent buffer front (FIG. 2A,lanes 4 and 6). However, the DOX-PhMV coat proteins (˜26 kDa) alsoshowed evidence of fluorescence, indicating that some DOX remainedassociated with the protein even under denaturing conditions within theelectrophoretic field (FIG. 2A, lane 6).

This may reflect hydrophobic interactions between DOX and aromatic aminoacids on the VLP internal surface. Anthracycline drugs such as DOXinteract with proteins such as human serum albumin and human α-1 acidglycoprotein via hydrophobic interactions that are stabilized byhydrogen bonds. We have previously proposed that DOX associates with PVXparticles via hydrophobic interactions, specifically involving π-πstacking between the benzene rings of DOX and nonpolar aromatic aminoacids such as phenylalanine, tyrosine and tryptophan. Likewise, DOX canalso interact via π-π stacking with other DOX molecules. After Coomassiestaining, the PS-PhMV and DOX-PhMV coat proteins appeared as two bandsrepresenting the monomer and a dimer, indicating some degree ofaggregation (FIG. 2A, lane 4 and 6). However inter-particle aggregationwas not apparent during FPLC and TEM analysis (see below).

Native agarose gel electrophoresis was used to confirm that PS and DOXwere associated with the VLPs and were not free in solution. Afterseparating the loaded particles, the gels were visualized under UVlight, stained with Coomassie, and imaged under white light (FIG. 2B).Both particles showed fluorescence under UV light, indicating stableassociation with PS and DOX. Free drug molecules were not detectedduring native electrophoresis (FIG. 2B, lanes 5 and 7). In the electricfield, the VLPs and both cargo molecules migrate towards the cathodebecause all three are positively charged (FIG. 2B, lane 4, 5 and 6, 7).Imaging the gels after Coomassie staining confirmed the presence ofnative PhMV, PS-PhMV and DOX-PhMV as single bands in the correspondinglanes (FIG. 2B, lane 1, 5 and 6). The co-migration of PhMV, PS-PhMV andDOX-PhMV particles indicated that the cargo molecules are encapsulatedby the VLPs, although additional surface association cannot be excluded.

Size exclusion chromatography (SEC) using FPLC and a Superose-6 columnshowed the typical elution profiles for intact drug-loaded VLPs,consistent with the elution profile of native PhMV (FIG. 2C; bottompanel). The FPLC profiles also indicated the co-elution of PS and DOX at450 nm and 496 nm, respectively, confirming the successful loading ofthese molecules. Finally, TEM analysis confirmed the integrity of theparticles after loading with PS or DOX. The TEM images clearly showedthat the approximately spherical structure of the wild-type particleswas unchanged after loading, and there was no significant change in sizefrom the wild-type particle diameter of 28-29 nm (FIG. 2D; bottompanel). The stability of the loaded VLPs was tested again by FPLC afterstorage for several weeks or months in KP buffer at 4° C. The elutionprofiles did not change after storage, confirming that the cargo wasstably encapsulated, and there was no evidence of particle aggregation.Infusion therefore appears to be a suitable approach for the loading ofcargo molecules into PhMV-based VLPs.

The Efficacy of Photodynamic Therapy Using PS-PhMV Particles Against PC3Cells

The delivery of PS-PhMV particles and their efficacy in vitro wereevaluated in PC-3 cells because efficient internalization had alreadybeen demonstrated in these cells (FIG. 4D) and photodynamic therapy hasa strong potential for the treatment of prostate cancer. To evaluate theefficacy of PS-PhMV compared to free PS, we incubated the cells witheach reagent for 8 h, with concentrations of PS ranging from 0.01 to 5.0PM. Treatment was induced by exposing the cells to white light, whereascontrols were kept in the dark. Unloaded VLPs were used as additionalcontrols, at the same protein concentration as the highest dose ofdrug-loaded particles. Cell viability was measured using an MTT assay toassess metabolic activity. We found that the efficacy of free PS(IC₅₀=0.05 PM) was not significantly affected by encapsulation: the IC₅₀value of the PS-PhMV particles was 0.03 μM (FIG. 6A). PS-PhMV controlsmaintained in the dark showed no evidence of cytotoxicity, nor did theunloaded VLPs, demonstrating the biocompatibility of the PhMV platformtechnology. The performance of the PS-PhMV particles was confirmed bysetting up a LIVE/DEAD cell viability assay in 24-well plates. As above,confluent PC3 cells were incubated for 8 h with 5.0 μM free PS orPS-PhMV (or corresponding controls), a concentration that achievedmaximum cytotoxicity in the MTT assay. After removing any remainingextracellular PS/PS-PhMV, white light was applied for 30 min. The cellswere incubated overnight in the dark, then stained with a combination ofcalcein-AM and ethidium homodimer-1 to detect living and dead cells,respectively (FIG. 6B). As above, PS and PS-PhMV controls maintained inthe dark remained 100% viable, as did untreated cells ad illuminatedcells exposed to unloaded VLPs. In contrast, illuminated cells exposedto 5.0 μM PS or PS-PhMV showed 0% viability.

These data are consistent with our previous reports in which CPMV or TMVwere used to deliver PS. Other examples include dual-surface modifiedbacteriophage MS2 capsids encapsulating PS and carrying an externalJurkat-specific aptamer, resulting in the selective killing of Jurkatleukemia T cells. Similarly, the simultaneous modification ofbacteriophage Qβ VLPs with a metalloporphyrin-based PS and a glycanligand achieved the specific targeting of CD22⁺ cells.

DOX-PhMV Delivery to Breast and Ovarian Cancer Cells

Finally, we investigated whether DOX retained its cytotoxic activity inthe context of DOX-PhMV particles by exposing A2780 ovarian cancer cellsand MDA-MB-231 breast cancer cells to a range of concentrations of freeDOX (0.1 to 10 μM) and equivalent concentrations of DOX in the contextof DOX-PhMV particles. We found that the cytotoxicity of free DOX wasnot significantly affected by encapsulation. The IC₅₀ value of theDOX-PhMV particles was 0.3 μM in the breast cancer cells and 0.04 μM inthe ovarian cancer cells, equivalent to the corresponding values for thefree drug (FIGS. 6C, D). In both cell lines, the unloaded VLPs showed notoxicity at the protein concentration corresponding to the highest doseof DOX-PhMV, confirming the biocompatible nature of the VLP deliveryplatform. Based on the cell uptake and imaging data, we propose thatPhMV is taken up by endocytosis and is trafficked to the lysosome wherethe protein carrier is degraded thus releasing the guest molecule (DOXin this case) which diffuses into the cytosol and kills the cell.

DOX is a highly potent drug used for cancer treatment but deliveryvehicles are required to overcome its dose-limiting toxicity towardshealthy cells. Non-viral and viral delivery systems are undergoingpre-clinical and clinical testing. Although each carrier system hasadvantages and disadvantages, VLPs are robust, monodisperse, easy andinexpensive to produce, biocompatible, biodegradable andnon-infectious—they are also readily engineered for the site-specificintroduction of new functionalities by genetic modification orbioconjugation, which can increase their solubility, reduce theirimmunogenicity, allow specific cell targeting, increase the efficiencyof internalization, and increase their potency. Several plant viruseshave been used to encapsulate DOX by infusion though gating or simplediffusion and caging mechanisms. For example, the depletion of divalentcations (Ca²⁺ and Mg²⁺) induces significant conformational changes inthe Red clover necrotic mosaic virus (RCNMV) capsid, leading to thereversible formation of pores that allow the ingress of dyes and DOXmolecules into the internal cavity, where they bind to the negativelycharged virus genome. RCNMV was thus loaded with DOX and armed withtargeting peptides as a multifunctional tool to target and deliver cargoto cancer cells. Another example is encapsulated DOX nanoconjugatestargeted to folate-expressing cancer cells in vivo using the Cucumbermosaic virus (CMV) platform. We have demonstrated that CPMV, PVX and TMVachieve the efficient delivery of DOX and targeted cell killing usingbioconjugation and encapsulation protocols.

Whereas the reports discussed above used replication-competent virusesthat retain their genomic RNA, VLP platforms lack an infectious genomeand thus offer a safer alternative.

In summary, we developed bioconjugation chemistries and infusionprotocols that enable the functionalization of PhMV-based VLPs, thusproviding multiple approaches to modify the behavior and properties ofthe corresponding particles. The PhMV-derived VLPs are stable andinexpensive to produce, allowing their development as nanocarriers forin vitro drug delivery applications. The physical stability andbatch-to-batch consistency are advantageous because the functionalizedVLPs remain stable for long periods in storage. The production ofPhMV-based VLPs could be scaled up by increasing the capacity ofbacterial fermentation, but even greater scalability could be achievedby expression in plants. PhMV does not replicate in mammals, but isbiocompatible and biodegradable, adding a layer of safety compared tomammalian virus-based systems.

From the above description of the invention, those skilled in the artwill perceive improvements, changes and modifications. Suchimprovements, changes and modifications within the skill of the art areintended to be covered by the appended claims. All references,publications, and patents cited in the present application are hereinincorporated by reference in their entirety.

1: A method of treating and/or detecting cancer tissue in a subjectcomprising administering to the subject a plurality of functionalizedTymovirus virus or Tymovirus virus-like particles (VLPs) loaded with orconjugated to one or more of a therapeutic agent, an imaging agent, or atargeting agent. 2: The method of claim 1, wherein the Tymovirus virusbelongs to the physalis mottle virus (PhMV) species. 3: The method ofclaim 1, wherein the Tymovirus virus or VLPs have been PEGylated. 4: Themethod of claim 1, wherein the Tymovirus virus or VLPs comprise animaging agent. 5: The method of claim 4, wherein the imaging agent is afluorescent molecule for fluorescent imaging or a chelated metal for MRIimaging. 6: The method of claim 5, wherein the Tymovirus virus or VLPsare administered at an effective amount, and further comprising the stepof imaging cancer tissue in the subject using an imaging devicesubsequent to administering the Tymovirus virus or VLPs. 7: The methodof claim 1, wherein the Tymovirus virus or VLPs comprise a therapeuticagent. 8: The method of claim 7, wherein the therapeutic agent is anantitumor agent. 9: The method of claim 8, wherein the antitumor agentis selected from doxorubicin and mitoxantrone. 10: The method of claim1, wherein the cancer tissue is prostate cancer, breast cancer, orovarian cancer tissue. 11: The method of claim 1, wherein thetherapeutic agent is a photodynamic therapeutic (PDT) photosensitizeragent. 12: The method of claim 11, the PDT agent selected from aporphyrin or a mettalloporphyrin compound. 13: The method of claim 12,wherein the porphyrin is a cationic zinc ethynylphenyl porphyrin. 14:The method of claim 1, wherein the one or more of a therapeutic agent,an imaging agent, or a targeting agent is directly conjugated to theTymovirus virus or VLPs. 15: The method of claim 1, wherein the one ormore of a therapeutic agent, an imaging agent, or a targeting agent isconjugated to the Tymovirus virus or VLPs via a linker. 16: The methodof claim 1, including multiple targeting agents, wherein the spacing andlocation of targeting agents on each Tymovirus virus or VLP iscontrolled to facilitate delivery, targeting, and/or therapeuticefficacy of the Tymovirus virus or VLP. 17-56. (canceled)